Anti-claudin 18.2 antibodies and uses thereof

ABSTRACT

Provided are antibodies or fragment thereof having binding specificity to the wild-type human claudin 18.2 (CLDN18.2) protein and also capable of binding the M149L mutant. By contrast, such antibodies and fragments do not or weakly bind to claudin 18.1 (CLDN18.1).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/176,820 filed on Feb. 16, 2021, which is a division of U.S.application Ser. No. 16/962,817 filed on Jul. 16, 2020, which is a U.S.National Stage Application under 35 U.S.C. 371 of InternationalApplication No. PCT/CN2019/087591, filed May 20, 2019, which claimspriority to International Application No. PCT/CN2018/087443, filed May18, 2018. The contents of each of the aforementioned are herebyincorporated by reference in their entirety into the present disclosure.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on May 19, 2019, is named271339_SEQ_ST25.txt and is 146,879 bytes in size.

BACKGROUND

Claudins are a family of proteins that form the important components ofthe tight cell junctions. They establish a paracellular barrier whichcontrols the flow of molecules between the cells. The proteins haveN-terminus and a C-terminus in the cytoplasm. Different claudins areexpressed on different tissues, their altered function has linked toformation of cancers of respective tissues. Claudin-1 is expressed incolon cancer, claudin-18 is expressed in gastric cancer, and claudin-10is expressed in hepatocellular carcinoma.

Claudin-18 has two isoforms, isoform 1 and isoform 2. Isoform 2 (Claudin18.2 or CLDN18.2) is a highly selective cell lineage marker. Claudin18.2's expression in normal tissues is strictly confined todifferentiated epithelial cells of the gastric mucosa, but it was absentfrom the gastric stem cell zone. Claudin 18.2 was retained on malignanttransformation and was expressed in a significant proportion of primarygastric cancers and its metastases. Frequently ectopic activation ofclaudin 18.2 was also found in pancreatic, esophageal, ovarian, and lungtumors. These data suggested that CLDN18.2 has highly restrictedexpression pattern in normal tissues, with frequent ectopic activationin a diversity of human cancers.

SUMMARY

Anti-claudin 18.2 antibodies are discovered herein that have highaffinity to both the wild-type claudin 18.2 and a common mutant, M149L.Such antibodies, therefore, have the unique advantage of being capableof targeting both the wild-type and the M149L mutant claudin 18.2protein. This advantage is important because a significant portion ofcancer patients harbor this common mutation. Moreover, the antibodies ofthe present disclosure do not bind to the other claudin 18 isoform,claudin 18.1 and thus are highly selective.

In accordance with one embodiment of the present disclosure, provided isan antibody or fragment thereof having binding specificity to awild-type human claudin 18.2 (CLDN18.2) protein, wherein the antibody orfragment further binds to a M149L mutant of the CLDN18.2 protein at anaffinity that is at least about 1% of the affinity to the wild-typeCLDN18.2 protein, and wherein the antibody or fragment further does notbind to a human wild-type claudin 18.1 (CLDN18.1) protein at an affinitythat is greater than about 1% of the affinity to the wild-type CLDN18.2protein. In some embodiments, the antibody or fragment thereof does notbind to the CLDN18.1 protein.

Also provided, in one embodiment, is an antibody or fragment thereofhaving binding specificity to a wild-type human claudin 18.2 (CLDN18.2)protein, wherein the binding between the antibody or fragment thereofand the wild-type CLDN18.2 protein involves amino acid residuescomprising: at least an amino acid residue selected from the groupconsisting of Y46, G48, L49, W50, C53, V54, R55, E56 and S58; and atleast an amino acid residue selected from the group consisting of Y169and G172, of the wild-type CLDN18.2 protein. In some embodiments, thebinding further involves W30 of the CLDN18.2 protein.

In some embodiments, the binding involves amino acid residuescomprising: W30; two or more amino acid residues selected from the groupconsisting of G48, L49, W50, C53, and E56; and at least an amino acidresidue selected from the group consisting of Y169 and G172, of thewild-type CLDN18.2 protein. In some embodiments, the binding involvesamino acid residues comprising W30, G48, L49, W50, C53, E56 and Y169 ofthe wild-type CLDN18.2 protein.

Methods and uses for the treatment of diseases and conditions are alsoprovided. In one embodiment, provided is a method of treating cancer ina patient in need thereof, comprising administering to the patient theantibody or fragment thereof of the present disclosure. In anotherembodiment, a method of treating cancer in a patient in need thereof isprovided, comprising: (a) treating a cell, in vitro, with the antibodyor fragment thereof of the present disclosure; and (b) administering thetreated cell to the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that the mouse sera from all mice after DNA immunisationhave high titration reacted with HEK293 cells transfected with CLD 18A2by flow cytometry, CLD 18A1 as negtive control.

FIG. 2 shows that the hybridoma supernatants can bind to HEK293 cellstransfected with human CLD18A2 by cell ELISA or flow cytometry.

FIG. 3 shows that the purified murine antibodies can bind to MKN45 cellstransfected with human CLD18A2 by flow cytometry with high EC50,compared with postive reference antibody.

FIG. 4 shows that the purified murine antibodies can bind to SU620 cellsendogenously expressing human CLD18A2 bearing M149L mutation by flowcytometry with high EC50, while the reference antibody did not.

FIG. 5 shows that the purified murine antibodies can bind to HEK293cells transfected with mouse CLD18A2 by flow cytometry with high EC50.

FIG. 6 shows that the purified murine antibodies can bind to HEK293cells transfected with cyno CLD18A2 by flow cytometry with high EC50.

FIG. 7 shows that the purified murine antibodies can bind to HEK293cells transfected with human CLD18A2 by flow cytometry with high EC50.

FIG. 8 shows that the chimeric antibodies can bind to MKN45 cellstransfected with human CLD18A2 by flow cytometry with high EC50,compared with positive reference antibody.

FIG. 9 shows that the chimeric antibodies can not bind to MKN45 cellstransfected with human CLD18A1 by flow cytometry.

FIG. 10 shows that humanized antibodies can bind to MKN45 cellstransfected with human CLD18A2 by flow cytometry with high EC50,compared with positive reference antibody.

FIG. 11 shows that the humanized antibodies cannot bind to MKN45 cellstransfected with human CLD18A1 by flow cytometry.

FIG. 12 shows that humanized antibodies with CDR mutation can bind toMKN45 cells transfected with human CLD18A2 by flow cytometry with highEC50, compared with positive reference antibody.

FIG. 13 shows that the humanized antibodies with CDR mutation cannotbind to MKN45 cells transfected with human CLD18A1 by flow cytometry.

FIG. 14 shows that the de-risked variants had potent binding to cellsurface CLD18A2.

FIG. 15 shows that certain mutations of CLD18A2 have significant effecton the indicated antibodies binding to HEK293 cells transfected thesemutants, suggesting that these amino acid residues constitute at leastpart of the epitope.

FIG. 16 shows that antibodies 4F11E2, 72C1B6A3 and 120B7B2 had superiorbinding in both claudin 18.2 high and low CHO-K1 cells, as compared to175D10.

FIG. 17 shows the potent ADCC testing results of 4F11E2, 72C1B6A3 and120B7B2 using 175D10 antibody as a reference.

FIG. 18 shows that the S239D/I332E versions of the 4F11E2, 72C1B6A3 and120B7B2 outperformed the 175D10 counterpart in the ADCC assays.

FIG. 19 shows that 4F11E2, 72C1B6A3 and 120B7B2 also had better ADCPeffects than 175D10.

DETAILED DESCRIPTION Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

The term “isolated” as used herein with respect to cells, nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs orRNAs, respectively, that are present in the natural source of themacromolecule. The term “isolated” as used herein also refers to anucleic acid or peptide that is substantially free of cellular material,viral material, or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. Moreover, an “isolated nucleic acid” is meant to includenucleic acid fragments which are not naturally occurring as fragmentsand would not be found in the natural state. The term “isolated” is alsoused herein to refer to cells or polypeptides which are isolated fromother cellular proteins or tissues. Isolated polypeptides is meant toencompass both purified and recombinant polypeptides.

As used herein, the term “recombinant” as it pertains to polypeptides orpolynucleotides intends a form of the polypeptide or polynucleotide thatdoes not exist naturally, a non-limiting example of which can be createdby combining polynucleotides or polypeptides that would not normallyoccur together.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, though preferably less than 25% identity, withone of the sequences of the present disclosure.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” toanother sequence means that, when aligned, that percentage of bases (oramino acids) are the same in comparing the two sequences. This alignmentand the percent homology or sequence identity can be determined usingsoftware programs known in the art, for example those described inAusubel et al. eds. (2007) Current Protocols in Molecular Biology.Preferably, default parameters are used for alignment. One alignmentprogram is BLAST, using default parameters. In particular, programs areBLASTN and BLASTP, using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalentpolynucleotides are those having the above-noted specified percenthomology and encoding a polypeptide having the same or similarbiological activity.

The term “an equivalent nucleic acid or polynucleotide” refers to anucleic acid having a nucleotide sequence having a certain degree ofhomology, or sequence identity, with the nucleotide sequence of thenucleic acid or complement thereof. A homolog of a double strandednucleic acid is intended to include nucleic acids having a nucleotidesequence which has a certain degree of homology with or with thecomplement thereof. In one aspect, homologs of nucleic acids are capableof hybridizing to the nucleic acid or complement thereof. Likewise, “anequivalent polypeptide” refers to a polypeptide having a certain degreeof homology, or sequence identity, with the amino acid sequence of areference polypeptide. In some aspects, the sequence identity is atleast about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects,the equivalent polypeptide or polynucleotide has one, two, three, fouror five addition, deletion, substitution and their combinations thereofas compared to the reference polypeptide or polynucleotide. In someaspects, the equivalent sequence retains the activity (e.g.,epitope-binding) or structure (e.g., salt-bridge) of the referencesequence.

Hybridization reactions can be performed under conditions of different“stringency”. In general, a low stringency hybridization reaction iscarried out at about 40° C. in about 10×SSC or a solution of equivalentionic strength/temperature. A moderate stringency hybridization istypically performed at about 50° C. in about 6×SSC, and a highstringency hybridization reaction is generally performed at about 60° C.in about 1×SSC. Hybridization reactions can also be performed under“physiological conditions” which is well known to one of skill in theart. A non-limiting example of a physiological condition is thetemperature, ionic strength, pH and concentration of Mg²⁺ normally foundin a cell.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching. The term “polymorphism” refers to the coexistence of morethan one form of a gene or portion thereof. A portion of a gene of whichthere are at least two different forms, i.e., two different nucleotidesequences, is referred to as a “polymorphic region of a gene”. Apolymorphic region can be a single nucleotide, the identity of whichdiffers in different alleles.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA,miRNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers. A polynucleotide can comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure can be impartedbefore or after assembly of the polynucleotide. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. The term also refers to bothdouble- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of this disclosure that is a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

As used herein, an “antibody” or “antigen-binding polypeptide” refers toa polypeptide or a polypeptide complex that specifically recognizes andbinds to an antigen. An antibody can be a whole antibody and any antigenbinding fragment or a single chain thereof. Thus the term “antibody”includes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule having biological activityof binding to the antigen. Examples of such include, but are not limitedto a complementarity determining region (CDR) of a heavy or light chainor a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework (FR) region, or any portion thereof, or at least one portionof a binding protein.

The terms “antibody fragment” or “antigen-binding fragment”, as usedherein, is a portion of an antibody such as F(ab′)₂, F(ab)₂, Fab′, Fab,Fv, scFv and the like. Regardless of structure, an antibody fragmentbinds with the same antigen that is recognized by the intact antibody.The term “antibody fragment” includes aptamers, spiegelmers, anddiabodies. The term “antibody fragment” also includes any synthetic orgenetically engineered protein that acts like an antibody by binding toa specific antigen to form a complex.

A “single-chain variable fragment” or “scFv” refers to a fusion proteinof the variable regions of the heavy (V_(H)) and light chains (V_(L)) ofimmunoglobulins. In some aspects, the regions are connected with a shortlinker peptide of ten to about 25 amino acids. The linker can be rich inglycine for flexibility, as well as serine or threonine for solubility,and can either connect the N-terminus of the V_(H) with the C-terminusof the V_(L), or vice versa. This protein retains the specificity of theoriginal immunoglobulin, despite removal of the constant regions and theintroduction of the linker. ScFv molecules are known in the art and aredescribed, e.g., in U.S. Pat. No. 5,892,019.

The term antibody encompasses various broad classes of polypeptides thatcan be distinguished biochemically. Those skilled in the art willappreciate that heavy chains are classified as gamma, mu, alpha, delta,or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g.,γ1-γ4). It is the nature of this chain that determines the “class” ofthe antibody as IgG, IgM, IgA IgG, or IgE, respectively. Theimmunoglobulin subclasses (isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgG₅,etc. are well characterized and are known to confer functionalspecialization. Modified versions of each of these classes and isotypesare readily discernable to the skilled artisan in view of the instantdisclosure and, accordingly, are within the scope of the instantdisclosure. All immunoglobulin classes are clearly within the scope ofthe present disclosure, the following discussion will generally bedirected to the IgG class of immunoglobulin molecules. With regard toIgG, a standard immunoglobulin molecule comprises two identical lightchain polypeptides of molecular weight approximately 23,000 Daltons, andtwo identical heavy chain polypeptides of molecular weight53,000-70,000. The four chains are typically joined by disulfide bondsin a “Y” configuration wherein the light chains bracket the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion.

Antibodies, antigen-binding polypeptides, variants, or derivativesthereof of the disclosure include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized, primatized, or chimericantibodies, single chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv), fragments comprising either aVK or VH domain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto LIGHT antibodies disclosed herein) Immunoglobulin or antibodymolecules of the disclosure can be of any type (e.g., IgG, IgE, IgM,IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2)or subclass of immunoglobulin molecule.

Light chains are classified as either kappa or lambda (K, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VK) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CK) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen-binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CK domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VK domain and VH domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the VH and VK chains (i.e.CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some instances,e.g., certain immunoglobulin molecules derived from camelid species orengineered based on camelid immunoglobulins, a complete immunoglobulinmolecule may consist of heavy chains only, with no light chains. See,e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen-binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen-binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen-binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops which connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen-binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable regionby one of ordinary skill in the art, since they have been preciselydefined (see “Sequences of Proteins of Immunological Interest,” Kabat,E., et al., U.S. Department of Health and Human Services, (1983); andChothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196:901-917 (1987), which are incorporated hereinby reference in their entireties. The CDR definitions according to Kabatand Chothia include overlapping or subsets of amino acid residues whencompared against each other. Nevertheless, application of eitherdefinition to refer to a CDR of an antibody or variants thereof isintended to be within the scope of the term as defined and used herein.The appropriate amino acid residues which encompass the CDRs as definedby each of the above cited references are set forth in the table belowas a comparison. The exact residue numbers which encompass a particularCDR will vary depending on the sequence and size of the CDR. Thoseskilled in the art can routinely determine which residues comprise aparticular CDR given the variable region amino acid sequence of theantibody.

Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).

In addition to table above, the Kabat number system describes the CDRregions as follows: CDR-H1 begins at approximately amino acid 31 (i.e.,approximately 9 residues after the first cysteine residue), includesapproximately 5-7 amino acids, and ends at the next tryptophan residue.CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includesapproximately 16-19 amino acids, and ends at the next arginine or lysineresidue. CDR-H3 begins at approximately the thirty third amino acidresidue after the end of CDR-H2; includes 3-25 amino acids; and ends atthe sequence W-G-X-G, where X is any amino acid. CDR-L1 begins atapproximately residue 24 (i.e., following a cysteine residue); includesapproximately 10-17 residues; and ends at the next tryptophan residue.CDR-L2 begins at approximately the sixteenth residue after the end ofCDR-L1 and includes approximately 7 residues. CDR-L3 begins atapproximately the thirty third residue after the end of CDR-L2 (i.e.,following a cysteine residue); includes approximately 7-11 residues andends at the sequence F or W-G-X-G, where X is any amino acid.

Antibodies disclosed herein may be from any animal origin includingbirds and mammals Preferably, the antibodies are human, murine, donkey,rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. Inanother embodiment, the variable region may be condricthoid in origin(e.g., from sharks).

As used herein, the term “heavy chain constant region” includes aminoacid sequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain constant region comprises at least one of: aCH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region)domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.For example, an antigen-binding polypeptide for use in the disclosuremay comprise a polypeptide chain comprising a CH1 domain; a polypeptidechain comprising a CH1 domain, at least a portion of a hinge domain, anda CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3domain; a polypeptide chain comprising a CH1 domain, at least a portionof a hinge domain, and a CH3 domain, or a polypeptide chain comprising aCH1 domain, at least a portion of a hinge domain, a CH2 domain, and aCH3 domain. In another embodiment, a polypeptide of the disclosurecomprises a polypeptide chain comprising a CH3 domain. Further, anantibody for use in the disclosure may lack at least a portion of a CH2domain (e.g., all or part of a CH2 domain). As set forth above, it willbe understood by one of ordinary skill in the art that the heavy chainconstant region may be modified such that they vary in amino acidsequence from the naturally occurring immunoglobulin molecule.

The heavy chain constant region of an antibody disclosed herein may bederived from different immunoglobulin molecules. For example, a heavychain constant region of a polypeptide may comprise a CH1 domain derivedfrom an IgG₁ molecule and a hinge region derived from an IgG3 molecule.In another example, a heavy chain constant region can comprise a hingeregion derived, in part, from an IgG₁ molecule and, in part, from anIgG₃ molecule. In another example, a heavy chain portion can comprise achimeric hinge derived, in part, from an IgG₁ molecule and, in part,from an IgG₄ molecule.

As used herein, the term “light chain constant region” includes aminoacid sequences derived from antibody light chain. Preferably, the lightchain constant region comprises at least one of a constant kappa domainor constant lambda domain.

A “light chain-heavy chain pair” refers to the collection of a lightchain and heavy chain that can form a dimer through a disulfide bondbetween the CL domain of the light chain and the CH1 domain of the heavychain.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the VH domain and is amino terminal to the hinge region ofan immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat et al., U.S. Dept. of Health and Human Services, “Sequences ofProteins of Immunological Interest” (1983). The CH2 domain is unique inthat it is not closely paired with another domain. Rather, two N-linkedbranched carbohydrate chains are interposed between the two CH2 domainsof an intact native IgG molecule. It is also well documented that theCH3 domain extends from the CH2 domain to the C-terminal of the IgGmolecule and comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al., J. Immunol161:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CK regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant disclosure)is obtained from a second species. In certain embodiments the targetbinding region or site will be from a non-human source (e.g. mouse orprimate) and the constant region is human.

As used herein, “percent humanization” is calculated by determining thenumber of framework amino acid differences (i.e., non-CDR difference)between the humanized domain and the germline domain, subtracting thatnumber from the total number of amino acids, and then dividing that bythe total number of amino acids and multiplying by 100.

By “specifically binds” or “has specificity to,” it is generally meantthat an antibody binds to an epitope via its antigen-binding domain, andthat the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope, via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.”

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of cancer.Beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sport, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, andso on.

As used herein, phrases such as “to a patient in need of treatment” or“a subject in need of treatment” includes subjects, such as mammaliansubjects, that would benefit from administration of an antibody orcomposition of the present disclosure used, e.g., for detection, for adiagnostic procedure and/or for treatment.

Anti-Claudin 18.2 Antibodies

The present disclosure provides anti-claudin 18.2 antibodies with highaffinity to both the wild-type claudin 18.2 and a common mutant, M149L(the SU620 cell endogenously expressing this mutation). To the bestknowledge of the inventors, all currently known anti-claudin 18.2proteins do not bind to this mutant. The antibodies of the presentdisclosure, therefore, have the unique advantage of being capable oftargeting both the wild-type and the M149L mutant claudin 18.2 protein.This advantage is important because a significant portion of cancerpatients harbor this common mutation. It is also worth noting that theantibodies of the present disclosure do not bind to the other claudin 18isoform, claudin 18.1 (or binds claudin 18.1 at a much lower affinity).

Experiments presented in the Examples revealed that certain amino acidsin the first and second extracellular fragments of the claudin 18.2protein are involved in or otherwise impact the binding of the presentlydisclosed antibodies to the protein. More specifically, three clustersof amino acid residues are shown to represent the epitope of theantibodies. The first cluster includes W30, which is in the first partof the first extracellular domain of the claudin 18.2 protein. Thesecond cluster, including N45, Y46, G48, L49, W50, C53, V54, R55, E56,S58, F60, E62, and C63, is also located in the first extracellulardomain. The third cluster, including Y169 and G172, on the other hand,are located at or close to the second extracellular domain.

The antibodies and fragments of the present disclosure exhibitedsuperior properties even when using a clinical candidate as a reference.175D10 (IMAB362) is currently undergoing phase III clinical trials fortreating gastric and gastroesophageal junction adenocarcinoma. Theinstant antibodies and fragment not only showed stronger bindingactivities, they also exhibited higher ADCC and ADCP activities undervarious different conditions, as compared to 175D10.

The improved properties of these new antibodies and fragments, it iscontemplated, are attributed to the higher binding specificity of theseantibodies and fragments as compared to those under development, such as175D10. For instance, 175D10's interaction with claudin 18.2 is strongacross the spectrum in FIG. 15, which includes strong binding to D28,Q33, N38 and V43, and then G59 and V79. The new antibodies, 4F11E2,72C1B6A3 and 120B7B2, by contrast, have higher specificity to W30 withinthe first half of the first extracellular domain, and higher specificityto G48 through E56 within the second half of the first extracellulardomain. The new antibodies also have slightly stronger binding to Y46and S58, which are also in the second half. Their binding to D28, Q33,N38, V43, G59 and V79 is considerably weaker (or not functional), whichlikely contributed to the improved ADCC and ADCP of the new antibodies.

Human Claudin 18.2 Sequence

Name Sequence (SEQ ID NO: 30) Human   1 MAVTACQGLG FVVSLIGIAG IIAATCM DQ W  ST Q DLYNNPV TAVF NY Q GLW C1audin 18.2  51 RS CVRE S SGF  T ECRGYFTLL GLPAMLQAVR ALMIVGIVLG AIGLLVSIFA (NP_001002026)101 LKCIRIGSME DSAKANMTLT SGIMFIVSGL CAIAGVSVFA NMLVTNFWMS151 TANMYTGMGG MVQTVQTR Y T F G AALFVGWV AGGLTLIGGV MMCIACRGLA201 PEETNYKAVS YHASGHSVAY KPGGFKASTG FGSNTKNKKI YDGGARTEDE251 VQSYPSKHDY V

In accordance with one embodiment of the present disclosure, provided isan antibody or fragment thereof having binding specificity to awild-type human claudin 18.2 (CLDN18.2) protein, wherein the antibody orfragment further binds to a M149L mutant of the CLDN18.2 protein. Insome embodiments, the antibody or fragment does not bind to a humanwild-type claudin 18.1 (CLDN18.1) protein, or does not bind CLDN18.1 atan affinity that is greater than about 1% of the affinity to thewild-type CLDN18.2 protein.

The binding affinity of an antibody or fragment to a protein can bemeasured with many methods known in the art. For examples, it can bemeasured cell-free assays with standalone CLDN18.1 or CLDN18.2 proteins.Preferably, however, the measurement is done with the CLDN18.1 orCLDN18.2 protein on a cell surface mimicking the actual bindingenvironment. Such binding assays are adequately exemplified in theexperimental examples.

In some embodiments, the antibodies or fragments thereof have a bindingaffinity to the M149L mutant that is at least 1%, or alternatively atleast 0.001%, 0.01%, 0.1%, 0.5%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the affinityto the wild-type CLDN18.2 protein.

In some embodiments, the antibodies or fragments thereof do not bindhuman CLDN18.1. In some embodiments, the antibodies or fragmentsthereof, as compared to binding to CLDN18.2, have a much weaker bindingto human CLDN18.1, e.g., not greater than 10%, 5%, 2%, 1%, 0.5%, 0.1%,0.05%, 0.01%, 0.005%, or 0.001%, without limitation.

As described above, the antibodies and fragments thereof of the presentdisclosure bind to the claudin 18.2 protein at an epitope that isdifferent from known antibodies (see FIG. 4; at least the referenceantibody interacts with M149 whereas the presently disclosed ones donot). In one embodiment, therefore, provided is an antibody or fragmentthereof having binding specificity to a wild-type human claudin 18.2(CLDN18.2) protein, wherein the binding between the antibody or fragmentthereof and the wild-type CLDN18.2 protein involves amino acid residuescomprising at least an amino acid residue selected from the groupconsisting of Y46, G48, L49, W50, C53, V54, R55, E56 and S58; and atleast an amino acid residue selected from the group consisting of Y169and G172, of the wild-type CLDN18.2 protein. In some embodiments, thebinding further involves one or more of W30 of the CLDN18.2 protein.

In some embodiments, the antibody or fragment thereof binds to N45 ofCLDN18.2. In some embodiments, the antibody or fragment thereof binds toY46 of CLDN18.2. In some embodiments, the antibody or fragment thereofbinds to G48 of CLDN18.2. In some embodiments, the antibody or fragmentthereof binds to L49 of CLDN18.2. In some embodiments, the antibody orfragment thereof binds to W50 of CLDN18.2. In some embodiments, theantibody or fragment thereof binds to C53 of CLDN18.2. In someembodiments, the antibody or fragment thereof binds to V54 of CLDN18.2.In some embodiments, the antibody or fragment thereof binds to R55 ofCLDN18.2. In some embodiments, the antibody or fragment thereof binds toE56 of CLDN18.2. In some embodiments, the antibody or fragment thereofbinds to E58 of CLDN18.2. In some embodiments, the antibody or fragmentthereof binds to F60 of CLDN18.2. In some embodiments, the antibody orfragment thereof binds to E62 of CLDN18.2. In some embodiments, theantibody or fragment thereof binds to C63 of CLDN18.2.

In some embodiments, the antibody or fragment thereof binds to at leasttwo amino acid residues selected from G48, L49, W50, C53, V54, R55, andE56. In some embodiments, the antibody or fragment thereof binds to atleast three amino acid residues selected from G48, L49, W50, C53, V54,R55, and E56. In some embodiments, the antibody or fragment thereofbinds to at least four amino acid residues selected from G48, L49, W50,C53, V54, R55, and E56. In some embodiments, the antibody or fragmentthereof binds to at least five amino acid residues selected from G48,L49, W50, C53, V54, R55, and E56.

In some embodiments, the antibody or fragment thereof binds to at leasttwo amino acid residues selected from Y46, G48, L49, W50, C53, V54, R55,E56 and S58. In some embodiments, the antibody or fragment thereof bindsto at least three amino acid residues selected from Y46, G48, L49, W50,C53, V54, R55, E56 and S58. In some embodiments, the antibody orfragment thereof binds to at least four amino acid residues selectedfrom Y46, G48, L49, W50, C53, V54, R55, E56 and S58. In someembodiments, the antibody or fragment thereof binds to at least fiveamino acid residues selected from Y46, G48, L49, W50, C53, V54, R55, E56and S58.

In some embodiments, the antibody or fragment thereof binds to at leastY169 of CLDN18.2. In some embodiments, the antibody or fragment thereofbinds to at least G172 of CLDN18.2. In some embodiments, the antibody orfragment thereof binds to at least two amino acid residues selected fromY169 and G172 of CLDN18.2.

In some embodiments, the binding involves amino acid residues comprisingW30; two, three, four, five or more amino acid residues selected fromthe group consisting of G48, L49, W50, C53, and E56; and at least anamino acid residue selected from the group consisting of Y169 and G172,of the wild-type CLDN18.2 protein. In some embodiments, the bindinginvolves amino acid residues comprising W30, G48, L49, W50, C53, E56 andY169 of the wild-type CLDN18.2 protein.

In some embodiments, the binding between the antibody or fragmentthereof and the wild-type CLDN18.2 protein involves at least Y46 and/orS58. In some embodiments, the binding between the antibody or fragmentthereof and the wild-type CLDN18.2 protein does not involve N38 or V43.In some embodiments, the binding between the antibody or fragmentthereof and the wild-type CLDN18.2 protein does not involve D28, Q33,N38, V43, G59 and V79, or have weaker bindings to one, two, three, fouror all of D28, Q33, N38, V43, G59 and V79.

The weaker bindings to these amino acids on CLDN18.2 may be as comparedto the other amino acids, such as G48, L49, W50, C53, V54, R55, E56. Insome embodiments, the comparison is to the binding at the same aminoacid to 175D10. For instance, the binding of the antibody or fragment ofthe present disclosure is weaker than that of 175D10(IMGT/2Dstructure-DB card No: 10473) to at least one, two, three, four,five or all of D28, Q33, N38, V43, G59 and V79.

In some embodiments, the antibody or fragment thereof does not bindM149L of the CLDN18.2 protein. In some embodiments, the antibody orfragment thereof binds to a M149L mutant of the CLDN18.2 protein.

In accordance with one embodiment of the present disclosure, provided isan antibody or fragment thereof that includes the heavy chain and lightchain variable domains with the CDR regions as shown in the CDRcombinations of Table A and Table A′.

TABLE A CDR combinations of tested antibodies CDRL2 Comb. CDRL1 (SEQ IDCDRL3 CDRH1 CDRH2 CDRH3 No. Antibody (SEQ ID NO:) NO:) (SEQ ID NO:)(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)  1 64G11B4 QSLLNSGNQRNY WAS (2)QNDYFYPFT (42) GYAFTNYL (59) INPGNGGS (77) ARIYYGNSFAY (31) (96)  265G8B8 QSLLNSGNLKNY WAS (2) QNVYIYPFT (43) GFSLTSYG (60) IWGDGNT (78)AKQGLYGHAMDY (32) (97)  3 56E8F10F4 QSLLNSGNQKNY WAS (2) QNDYYFPFT (44)GFTFNSFG (61) ISGGSNTI (79) TRLALGNAMDY (1) (98)  4 54A2C4 QSLLNGGNQKNYGAS QNDLYYPWT (45) GFTFNTNA (62) IRSKSNNYAT (80) VSGAYYGNSKAFDY (33)(39) (99)  5 54A2C4′ QSLLNGGNQKNY GAS QNDLYYPWT (45) GYAFTNYL (59)INPGNGGSN (81) ARIYYGNSFAY (33) (39) (96)  6 54A2C4″ QSLLNGGNQKNY GASQNDLYYPWT (45) GYTFPTYS (63) INPSTIYT (82) AREGYGRGNAMDY (33) (39) (100) 7 44F6B11 QSLLNSGNQKKY WAS (2) QNGYSYPFT (46) GFTFSNYG (64)FSYGDSHN (83) ARFGRGNTMDY (34) (101)  8 15C2B7 QSLLNSGNQKNY WAS (2)QNNYYFPLT (47) GYTFTNYG (65) INANTGEP (84) ARLTRGNSFDY (1) (102)  920F1E10 QSLFNSGNQRNY WAS (2) QNVYSYPLT (48) GYTFTKYG (66) ISTNTGEP (85)ARLVRGNSFDF (35) (103) 10 72C1B6A3 QSLLNSGNQKNY RAS (7) QNDYIYPYT (8)GYTFTTYP (9) FHPYNDDT (10) ARRAYGYPYAMDY (1) (11) 11 58G2C2 QSLLNSGNQKNYWAF QNSYSYPFT (49) GYAFTNYL (59) INPGRSGT (86) ARTRYGGNAMDY (1) (40)(104) 12 101C4F12 QSLLNSGNQRNY WSS QNNFIYPLT (50) GFSLSSYG (67)IWAGGST (87) ARSLYGNSLDS (31) (41) (105) 13 103A10B2 MSLFNSGNQKSYWAS (2) HNDYIYPLT (51) GLSLTSFG (68) IWAGGST (87) ARSLYGNSFDY (36) (106)14 78E8G9G6 QSLLNSGNQKNY WAS (2) QNSYSYPFT (49) GFSLISYG (69)IWAGGRT (88) ARDRYGGNSLDY (1) (107) 15 4F11E2 QSLLNSGNRKNY WAS (2)QNAYSYPFT (13) GFTFSTFG (14) ITSGNSPI (15) ARSSYYGNSMDY (12) (16) 1610G7G11 QSLFNSGNQRNY WAS (2) QNAYYFPFT (19) GFSLNTYG (70) MLSDGNT (89)ARHKAYGNAMDY (35) (108) 17 12F1F4 QSLFNSGNQRNY WSS QNNYYYPFT (52)GFSLINYG (71) IWGDGNT (78) AKVGRGNAMDH (35) (41) (109) 18 78C10B6G4QSLLNSGNQKNY RAS (7) QNDYIYPYT (8) GFSLINYG (71) IRGDGNT (90)AKVGRGNAMDH (1) (109) 19 119G11D9 QSLFNSGNQKNY WAS (2) QNAYYYPLT (53)GYTFTGFL (72) INPYNDGT (5) ARLDYGNAMDY (37) (110) 20 113G12E5E6QSLLNSGNQKNY WAS (2) QNAYFYPCT (54) DFSLTKYG (73) IWTGGNT (91)ARNGYYGNAMDY (1) (111) 21 116A8B7 QSLFNSGNQRNY WAS (2) QNAYYYPLT (53)GYTFTGFL (72) INPYNDGT (5) GRLDYGNAMDY (35) (112) 22 105F7G12QSLLNSGNQKNY WAS (2) QNAYFYPCT (54) DFSLTKYG (73) IWTGGNT (91)ARNGYYGNAMDY (1) (111) 23 84E9E12 QSVFNSGNQKNY WAS (2) QNDYYFPLT (55)GYSITSGYF ISYDGSN (92) ASFRFFAY(113) (38) (74) 24 103F4D4 QSLLNGGNQKNYWAS (2) QNAYFYPFT (56) GYTFPTYS (63) INPSTIYT (82) AREGYGRGNAMDY (33)(100) 25 110C12B6 QSLFNSGNQRNY WAS (2) QNAYYYPLT (53) GYTFTGFL (72)INPYNDGT (5) GRLDYGNAMDY (35) (112) 26 85H12E8 QSLLNSGNQRNY WAS (2)QNAYFYPFT (56) GFSLSNYG (75) IWAGGNT (93) ARHGYGKGNAMDN (31) (114) 27103H2B4 QSLLNSGNQKNY WAS (2) QNNYFYPLT (57) GYSFTNFL (76) INPTNGRT (94)ARIYYGNSMDY (1) (115) 28 103F6D3 QSLLNGGNQKNY WAS (2) QNAYFYPFT (56)GYTFPTYS (63) INPNTIYT (95) AREGYGRGNAMDY (33) (100) 29 113E12F7QSLFNSGNQKNY WAS (2) QNNYIYPLA (58) GFSLSSYG (67) IWAGGST (87)ARSLYGNSFDH (37) (116) 30 120137132 QSLLNSGNQKNY WAS (2) QNGYYFPFT (3)GYTFTGYI (4) INPYNDGT (5) ARAYFGNSFAY (1) (6) 31 111612D11 QSLFNSGNQRNYWAS (2) QNNYIYPLA (58) GFSLTSYG (60) IWAGGST (87) ARSLYGNSFDH (35) (116)32 111E7E2 QSLFNSGNQKNY WAS (2) QNNYIYPLA (58) GFSLTSYG (60)IWAGGST (87) ARSLYGNSFDH (37) (116) 33 100F4G12 QSLLNSGNQRNY WAS (2)QNAYYYPLT (53) GYTFTGFL (72) INPYNDGT (5) ARLDYGNAMDY (31) (110)

TABLE A′ CDR combination of tested antibodies (Kabat numbering) CDRL2CDRL3 CDRH1 CDRH3) Comb. CDRL1 (SEQ (SEQ (SEQ CDRH2 SEQ No. Antibody(SEQ ID NO:) ID NO:) ID NO:) ID NO:) (SEQ ID NO:) ID NO:)  1 64G11B4KSSQSLLNSGNQRNYLT WASTRES QNDYFYPFT NYLLE EINPGNGGSNYNEKFKG IYYGNSFAY(208) (227) (42) (234) (255) (281)  2 65G8B8 KSSQSLLNSGNLKNYLT WASTRESQNVYIYPFT SYGVS VIWGDGNTIYHSALKS QGLYGHAMDY (209) (227) (43) (235) (256)(282)  3 56E8F10F4 KSSQSLLNSGNQKNYLT WASTRES QNDYYFPFT SFGMNFISGGSNTIHYLDTVKG LALGNAMDY (210) (227) (44) (236) (257) (283)  4 54A2C4KSSQSLLNGGNQKNYLA GASTRES QNDLYYPWT TNAMN RIRSKSNNYATYYADSVKDGAYYGNSKAFDY (211) (228) (45) (237) (258) (284)  5 54A2C4′KSSQSLLNGGNQKNYLA GASTRES QNDLYYPWT NYLLE EINPGNGGSNYNEKFKG IYYGNSFAY(211) (228) (45) (234) (255) (281)  6 54A2C4″ KSSQSLLNGGNQKNYLA GASTRESQNDLYYPWT TYSIH YINPSTIYTNYNQKFKY EGYGRGNAMDY (211) (228) (45) (238)(259) (285)  7 44F6B11 KSNQSLLNSGNQKKYLT WASTRES QNGYSYPFT NYGMSTFSYGDSHNYYSDSVKG FGRGNTMDY (212) (227) (46) (239) (260) (286)  8 15C2B7KSSQSLLNSGNQKNYLT WASTRES QNNYYFPLT NYGMN WINANTGEPTYAEEFKG LTRGNSFDY(210) (227) (47) (240) (261) (287)  9 20F1E10 KSSQSLFNSGNQRNYLT WASTRESQNVYSYPLT KYGMN WISTNTGEPTYAEEFKG LVRGNSFDF (213) (227) (48) (241) (262)(288) 10 72C1B6A3 KSSQSLLNSGNQKNYLT RASSRES QNDYIYPYT TYPIENFHPYNDDTKYNEKFKG RAYGYPYAMDY (210) (229) (8) (242) (263) (289) 1158G2C2 KSSQSLLNSGNQKNYLT WAFTRES QNSYSYPFT NYLIE VINPGRSGTNYNEKFKGTRYGGNAMDY (210) (230) (49) (243) (264) (290) 12 101C4F12KSSQSLLNSGNQRNYLT WSSTRDS QNNFIYPLT SYGVH VIWAGGSTNYDSALMS SLYGNSLDS(208) (231) (50) (244) (265) (291) 13 103A10B2 RSSMSLFNSGNQKSYLS WASTRDSHNDYIYPLT SFGVH VIWAGGSTNYNSALMS SLYGNSFDY (214) (232) (51) (245) (266)(292) 14 78E8G9G6 RSIQSLLNSGNQKNYLS WASTRES QNSYSYPFT SYGVHVIWAGGRTNYNSALMS DRYGGNSLDY (215) (227) (49) (244) (267) (293) 15 4F11E2RSSQSLLNSGNRKNYLT WASTRES QNAYSYPFT TFGMH YITSGNSPIYFTDTVKG SSYYGNSMDY(216) (227) (13) (246) (268) (294) 16 10G7G11 KSSQSLFNSGNQRNYLT WASTRESQNAYYFPFT TYGVH VMLSDGNTVYNSSLKS HKAYGNAMDY (213) (227) (19) (247) (269)(295) 17 12F1F4 KSSQSLFNSGNQRNYLT WSSTRES QNNYYYPFT NYGVSVIWGDGNTNYQSALRS VGRGNAMDH (213) (233) (52) (248) (270) (296) 1878C10B6G4 KSSQSLLNSGNQKNYLT RASSRES QNDYIYPYT NYGVS VIRGDGNTNYQSALRSVGRGNAMDH (210) (229) (8) (248) (271) (296) 19 119G11D9RSTQSLFNSGNQKNYLT WASTRES QNAYYYPLT GFLMH YINPYNDGTKYSEKFKG LDYGNAMDY(217) (227) (53) (249) (272) (297) 20 113G12E5E6 KPSQSLLNSGNQKNYLAWASTRES QNAYFYPCT KYGVH VIWTGGNTDYNPALIP NGYYGNAMDY (218) (227) (54)(250) (273) (298) 21 116A8B7 RSTQSLFNSGNQRNYLT WASTRES QNAYYYPLT GFLMHYINPYNDGTKYSEKFKG LDYGNAMDY (219) (227) (53) (249) (272) (297) 22105F7G12 KSSQSLLNSGNQKNYLA WASTRES QNAYFYPCT KYGVH VIWTGGNTDYNPALIPNGYYGNAMDY (220) (227) (54) (250) (273) (298) 23 84E9E12KSSQSVFNSGNQKNYLT WASTRES QNDYYFPLT SGYFW YISYDGSNNYNPSLKN FRFFAY (221)(227) (55) (251) (274) (299) 24 103F4D4 RSSQSLLNGGNQKNYLT WASTRESQNAYFYPFT TYSIH YINPSTIYTNYNQKFKY EGYGRGNAMDY (222) (227) (56) (238)(259) (285) 25 110C12B6 RSTQSLFNSGNQRNYLT WASTRES QNAYYYPLT GFLMHYINPYNDGTKYSEKFKG LDYGNAMDY (219) (227) (53) (249) (272) (297) 2685H12E8 KSSQSLLNSGNQRNYLS WASTRES QNAYFYPFT NYGVS VIWAGGNTNYNSALMSHGYGKGNAMDN (223) (227) (56) (248) (275) (300) 27 103H2B4KSSQSLLNSGNQKNYLT WASTRES QNNYFYPLT NFLTH EINPTNGRTYYNEKFKR IYYGNSMDY(210) (227) (57) (252) (276) (301) 28 103F6D3 RSSQSLLNGGNQKNYLT WASTRESQNAYFYPFT TYSIH YINPNTIYTNYNQKFKY EGYGRGNAMDY (222) (227) (56) (238)(277) (285) 29 113E12F7 KSSQSLFNSGNQKNYLT WASTRES QNNYIYPLA SYGVHVIWAGGSTNYDSALMS SLYGNSFDH (224) (227) (58) (244) (265) (302) 30 1206762KSSQSLLNSGNQKNYLT WASTRES QNGYYFPFT GYIIQ FINPYNDGTKYNEQFKG AYFGNSFAY(210) (227) (3) (253) (278) (303) 31 111612D11 RSSQSLFNSGNQRNYLT WASTRESQNNYIYPLA SYGVH VIWAGGSTNYDSTLMS SLYGNSFDH (225) (227) (58) (244) (279)(302) 32 111E7E2 KSSQSLFNSGNQKNYLT WASTRES QNNYIYPLA SYGAHVIWAGGSTNYDSALMS SLYGNSFDH (224) (227) (58) (254) (265) (302) 33100F4G12 KSTQSLLNSGNQRNYLT WASTRES QNAYYYPLT GFLMH YINPYNDGTKYSERFKGLDYGNAMDY (226) (227) (53) (249) (280) (297)

TABLE B CDRs of 120B7B2 Sequence De-Risked Versions CDR (SEQ ID NO:)(SEQ ID NO:) CDRL1 QSLLNSGNQKNY (1) QSLLN A GNQKNY (17) QSLL ESGNQKNY (18) CDRL2 WAS (2) CDRL3 QNGYYFPFT (3) QN A YYFPFT (19) Q EGYYFPFT (20) CDRH1 GYTFTGYI (4) CDRH2 INPYNDGT (5) INPYND D T (21) CDRH3ARAYFGNSFAY (6) ARAYFGN A FAY (22)

TABLE C CDRs of 72C1B6A3 Sequence De-Risked Versions CDR (SEQ ID NO:)(SEQ ID NO:) CDRL1 QSLLNSGNQKNY (1) QSLLN A GNQKNY (17) QSLL ESGNQKNY (18) CDRL2 RAS (7) CDRL3 QNDYIYPYT (8) CDRH1 GYTFTTYP (9) CDRH2FHPYNDDT (10) CDRH3 ARRAYGYPYAMDY (11)

TABLE D CDRs of 4F11E2 Sequence De-Risked Versions CDR (SEQ ID NO:)(SEQ ID NO:) CDRL1 QSLLNSGNRKNY (12) QSLL E SGNRKNY (23) QSLLN AGNRKNY (24) CDRL2 WAS (2) CDRL3 QNAYSYPFT (13) CDRH1 GFTFSTFG (14) CDRH2ITSGNSPI (15) ITSG Q SPI (25) ITSG E SPI (26) CDRH3 ARSSYYGNSMDY (16)ARSSYYG Q SMDY (27) ARSSYYG E SMDY (28) ARSSYYGN A MDY (29)

TABLE B′ CDRs of 120B7B2 (Kabat numbering) Sequence De-Risked VersionsCDR (SEQ ID NO:) (SEQ ID NO:) CDRL1 KSSQSLLNSGNQKNYLT KSSQSLLN AGNQKNYLT (304) (210) KSSQSLL E SGNQKNYLT (305) CDRL2 WASTRES (227) CDRL3QNGYYFPFT (3) QN A YYFPFT (19) Q E GYYFPFT (20) CDRH1 GYIIQ (253) CDRH2FINPYNDGTKYNEQFKG FINPYND D TKYNEQFKG (306) (278) CDRH3 AYFGNSFAY (303)AYFGN A FAY (307)

TABLE C′ CDRs of 72C1B6A3 (Kabat numbering) Sequence De-Risked VersionsCDR (SEQ ID NO:) (SEQ ID NO:) CDRL1 KSSQSLLNSGNQKNYLT KSSQSLLN AGNQKNYLT (304) (210) KSSQSLL E SGNQKNYLT (305) CDRL2 RASSRES (229) CDRL3QNDYIYPYT (8) CDRH1 TYPIE (242) CDRH2 NFHPYNDDTKYNEKFKG (263) CDRH3RAYGYPYANIDY (289)

TABLE D′ CDRs of 4F11E2 (Kabat numbering) Sequence De-Risked VersionsCDR (SEQ ID NO:) (SEQ ID NO:) CDRL1 RSSQSLLNSGNRKNYLT RSSQSLL ESGNRKNYLT (308) (216) RSSQSLLN A GNRKNYLT (309) CDRL2 WASTRES (227)CDRL3 QNAYSYPFT (13) CDRH1 TFGMH (246) CDRH2 YITSGNSPIYFTDTVKG YITSG QSPIYFTDTVKG (310) (268) YITSG E SPIYFTDTVKG (311) CDRH3 SSYYGNSMDY (294)SSYYG Q SMDY (312) SSYYG E SMDY (313) SSYYGN A MDY (314)

The antibodies that contained these CDR regions, whether mouse,humanized or chimeric, had potent claudin 18.2 binding and inhibitoryactivities. As shown in Examples 11 and 12, certain residues within theCDR can be modified to retain or improve the property or reduce theirpotential to have post-translational modifications (PTMs). Such modifiedCDR can be referred to as affinity matured or de-risked CDRs.

Non-limiting examples of de-risked CDRs are provided in Tables B-D andB′-D′, in the third columns. Affinity matured ones can include thosehaving one, two or three amino acid addition, deletion and/orsubstitutions. In some embodiments, the substitutions can beconservative substitutions.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in an immunoglobulin polypeptide is preferably replaced withanother amino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members.

Non-limiting examples of conservative amino acid substitutions areprovided in the table below, where a similarity score of 0 or higherindicates conservative substitution between the two amino acids.

TABLE E Amino Acid Similarity Matrix C G P S A T D E N Q H K R V M I L FY W W −8 −7 −6 −2 −6 −5 −7 −7 −4 −5 −3 −3 2 −6 −4 −5 −2 0 0 17 Y 0 −5 −5−3 −3 −3 −4 −4 −2 −4 0 −4 −5 −2 −2 −1 −1 7 10 F −4 −5 −5 −3 −4 −3 −6 −5−4 −5 −2 −5 −4 −1 0 1 2 9 L −6 −4 −3 −3 −2 −2 −4 −3 −3 −2 −2 −3 −3 2 4 26 I −2 −3 −2 −1 −1 0 −2 −2 −2 −2 −2 −2 −2 4 2 5 M −5 −3 −2 −2 −1 −1 −3−2 0 −1 −2 0 0 2 6 V −2 −1 −1 −1 0 0 −2 −2 −2 −2 −2 −2 −2 4 R −4 −3 0 0−2 −1 −1 −1 0 1 2 3 6 K −5 −2 −1 0 −1 0 0 0 1 1 0 5 H −3 −2 0 −1 −1 −1 11 2 3 6 Q −5 −1 0 −1 0 −1 2 2 1 4 N −4 0 −1 1 0 0 2 1 2 E −5 0 −1 0 0 03 4 D −5 1 −1 0 0 0 4 T −2 0 0 1 1 3 A −2 1 1 1 2 S − 1 1 1 P −3 −1 6 G−3 5 C 12

TABLE F Conservative Amino Acid Substitutions For Amino AcidSubstitution With Alanine D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys ArginineD-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-GluGln, D-Gln Aspartic Acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-GlnCysteine D-Cys, S—Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser GlutamineD-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid D-Glu, D-Asp,Asp, Asn, D-Asn, Gln, D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, β-AlaIsoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val,Met, D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Orn, D-OrnMethionine D-Met, S—Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-ValPhenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline D-ProSerine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys Threonine D-Thr, Ser,D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr, Phe, D-Phe, His,D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

In one embodiment, therefore, provided is an antibody or fragmentthereof having binding specificity to a wild-type human claudin 18.2(CLDN18.2) protein, wherein the antibody or fragment thereof comprises alight chain variable region comprising light chain complementaritydetermining regions CDRL1, CDRL2, and CDRL3 and a heavy chain variableregion comprising heavy chain complementarity determining regions CDRH1,CDRH2, and CDRH3, and wherein the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, andCDRH3 are selected from combinations 1-33 of Table A or Table A′ or eachof the combinations 1-33 in which one or more of the CDRL1, CDRL2,CDRL3, CDRH1, CDRH2, and CDRH3 each includes one, two, or three aminoacid addition, deletion, conservative amino acid substitution or thecombinations thereof.

In some embodiments, an anti-CLDN18.2 antibody or fragment is providedthat includes CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3, each ofwhich is selected from Table A or Tables B-D. For instance, provided isan antibody or fragment thereof having binding specificity to awild-type human claudin 18.2 (CLDN18.2) protein, wherein the antibody orfragment thereof comprises a light chain variable region comprisinglight chain complementarity determining regions CDRL1, CDRL2, and CDRL3and a heavy chain variable region comprising heavy chain complementaritydetermining regions CDRH1, CDRH2, and CDRH3, and wherein: the CDRL1comprises an amino acid sequence selected from the group of SEQ ID NO:1,12 and 31-38, or comprises an amino acid sequence derived anyone of SEQID NO:1, 12 and 31-38 by one, two, or three amino acid addition,deletion, amino acid substitution; the CDRL2 comprises an amino acidsequence selected from the group of SEQ ID NO:2, 7 and 39-41, orcomprises an amino acid sequence derived anyone of SEQ ID NO:2, 7 and39-41 by an amino acid addition, deletion, amino acid substitution; theCDRL3 comprises an amino acid sequence selected from the group of SEQ IDNO:3, 8, 13, 19 and 42-58, or comprises an amino acid sequence derivedanyone of SEQ ID NO: 3, 8, 13, 19 and 42-58 by one, two, or three aminoacid addition, deletion, amino acid substitution; the CDRH1 comprises anamino acid sequence selected from the group of SEQ ID NO:4, 9, 14 and59-76, or comprises an amino acid sequence derived anyone of SEQ IDNO:4, 9, 14 and 59-76 by one, two, or three amino acid addition,deletion, amino acid substitution; the CDRH2 comprises an amino acidsequence selected from the group of SEQ ID NO:5, 10, 15 and 77-95, orcomprises an amino acid sequence derived anyone of SEQ ID NO:5, 10, 15and 77-95 by one, two, or three amino acid addition, deletion, aminoacid substitution; and the CDRH3 comprises an amino acid sequenceselected from the group of SEQ ID NO:6, 11, 16 and 96-116, or comprisesan amino acid sequence derived anyone of SEQ ID NO:6, 11, 16 and 96-116by one, two, or three amino acid addition, deletion, amino acidsubstitution.

In some embodiments, the CDRL1 comprises an amino acid sequence selectedfrom the group of SEQ ID NO:1, 12, 17-18, 23-24 and 31-38; the CDRL2comprises an amino acid sequence selected from the group of SEQ ID NO:2,7 and 39-41; the CDRL3 comprises an amino acid sequence selected fromthe group of SEQ ID NO:3, 8, 13, 19-20 and 42-58; the CDRH1 comprises anamino acid sequence selected from the group of SEQ ID NO:4, 9, 14 and59-76; the CDRH2 comprises an amino acid sequence selected from thegroup of SEQ ID NO:5, 10, 15, 21, 25-26 and 77-95; and the CDRH3comprises an amino acid sequence selected from the group of SEQ ID NO:6,11, 16, 22, 27-29 and 96-116.

In some embodiments, an anti-CLDN18.2 antibody or fragment is providedthat includes CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3, each ofwhich is selected from Table A′ or Tables B′-D′. For instance, providedis an antibody or fragment thereof having binding specificity to awild-type human claudin 18.2 (CLDN18.2) protein, wherein the antibody orfragment thereof comprises a light chain variable region comprisinglight chain complementarity determining regions CDRL1, CDRL2, and CDRL3and a heavy chain variable region comprising heavy chain complementaritydetermining regions CDRH1, CDRH2, and CDRH3, and wherein: the CDRL1comprises an amino acid sequence selected from the group of SEQ IDNO:208-226, or comprises an amino acid sequence derived anyone of SEQ IDNO:208-226 by one, two, or three amino acid addition, deletion, aminoacid substitution; the CDRL2 comprises an amino acid sequence selectedfrom the group of SEQ ID NO:227-233, or comprises an amino acid sequencederived anyone of SEQ ID NO:227-233 by an amino acid addition, deletion,amino acid substitution; the CDRL3 comprises an amino acid sequenceselected from the group of SEQ ID NO:3, 8, 13, 19 and 42-58, orcomprises an amino acid sequence derived anyone of SEQ ID NO: 3, 8, 13,19 and 42-58 by one, two, or three amino acid addition, deletion, aminoacid substitution; the CDRH1 comprises an amino acid sequence selectedfrom the group of SEQ ID NO:234-254, or comprises an amino acid sequencederived anyone of SEQ ID NO:234-254 by one, two, or three amino acidaddition, deletion, amino acid substitution; the CDRH2 comprises anamino acid sequence selected from the group of SEQ ID NO:255-280, orcomprises an amino acid sequence derived anyone of SEQ ID NO:255-280 byone, two, or three amino acid addition, deletion, amino acidsubstitution; and the CDRH3 comprises an amino acid sequence selectedfrom the group of SEQ ID NO:281-303, or comprises an amino acid sequencederived anyone of SEQ ID NO:281-303 by one, two, or three amino acidaddition, deletion, amino acid substitution.

In some embodiments, the CDRL1 comprises an amino acid sequence selectedfrom the group of SEQ ID NO:208-226, 304-305 and 308-309; the CDRL2comprises an amino acid sequence selected from the group of SEQ IDNO:227-233; the CDRL3 comprises an amino acid sequence selected from thegroup of SEQ ID NO:3, 8, 13, 19, 20 and 42-58; the CDRH1 comprises anamino acid sequence selected from the group of SEQ ID NO:234-254; theCDRH2 comprises an amino acid sequence selected from the group of SEQ IDNO:255-280, 306, 310 and 311; and the CDRH3 comprises an amino acidsequence selected from the group of SEQ ID NO:281-303, 307, and 312-314.

The antibody 120B7B2 has been demonstrated to be potent inhibitor ofclaudin 18.2. Its CDR sequences, along with a few de-risked versions,are provided in Table B. In one embodiment, the present disclosureprovides an antibody or fragment thereof having binding specificity to awild-type human claudin 18.2 (CLDN18.2) protein, wherein the antibody orfragment thereof comprises a light chain variable region comprisinglight chain complementarity determining regions CDRL1, CDRL2, and CDRL3and a heavy chain variable region comprising heavy chain complementaritydetermining regions CDRH1, CDRH2, and CDRH3, and wherein: the CDRL1comprises the amino acid sequence of QSLLNSGNQKNY (SEQ ID NO:1),QSLLNAGNQKNY (SEQ ID NO:17) or QSLLESGNQKNY (SEQ ID NO:18) or an aminoacid sequence having one, two or three amino acid substitution from SEQID NO:1, 17 or 18, the CDRL2 comprises the amino acid sequence of WAS(SEQ ID NO:2) or an amino acid sequence having one or two amino acidsubstitution from SEQ ID NO:2, the CDRL3 comprises the amino acidsequence of CQNGYYFPFT (SEQ ID NO:3), QNAYYFPFT (SEQ ID NO:19) orQEGYYFPFT (SEQ ID NO:20) or an amino acid sequence having one, two orthree amino acid substitution from SEQ ID NO:3, 19 or 20, the CDRH1comprises the amino acid sequence of GYTFTGYI (SEQ ID NO:4) or an aminoacid sequence having one, two or three amino acid substitution from SEQID NO:4, the CDRH2 comprises the amino acid sequence of INPYNDGT (SEQ IDNO:5) or INPYNDDT (SEQ ID NO:21) or an amino acid sequence having one,two or three amino acid substitution from SEQ ID NO:5 or 21, and theCDRH3 comprises the amino acid sequence of ARAYFGNSFAY (SEQ ID NO:6) orARAYFGNAFAY (SEQ ID NO:22) or an amino acid sequence having one, two orthree amino acid substitution from SEQ ID NO:6 or 22.

It is interesting to note (see Table A) that the CDRs from differentantibody share great homology. It is then contemplated that eachcorresponding CDR can be interchanged without greatly impacting theantibody or fragment's binding affinity or activity. Alternatively, eachparticular amino acid in a CDR can be substituted with another aminoacid present in a corresponding CDR from a different antibody. In someembodiments, therefore, the following tables summarize some feasiblesubstitutions based on sequence alignments.

TABLE G1 Example substitutions of amino acid residues in SEQ ID NO: 1Kabat numbering Residue Substitution 27 Q M 29 L V 30 L F 30B S G 30E QL or R 30F K R 31 N K or S

TABLE G2 Example substitutions of amino acid residues in SEQ ID NO: 2Kabat numbering Residue Substitution 50 W G or R 51 A S 52 S F

TABLE G3 Example substitutions of amino acid residues in SEQ ID NO: 3Kabat numbering Residue Substitution 89 Q H 91 G A, D, N, S or V 92 Y For L 93 Y F, I or S 94 F Y 96 F C, L, W, or Y 97 T A

TABLE G4 Example substitutions of amino acid residues in SEQ ID NO: 4Kabat numbering Residue Substitution 26 G D 27 Y F or L 28 T A or S 29 FI or L 30 T I, N, P or S 31 G K, N, S or T 32 Y F, G or N 33 I A, G, L,P, S or Y

TABLE G5 Example substitutions of amino acid residues in SEQ ID NO: 5Kabat numbering Residue Substitution 51 I F or M 52 N H, I, L, R, S, Tor W 52A P A, G, S, T or Y 53 Y D, G, K, N, S or T 54 N D, G, R, S or T55 D G, R, S or T 56 G D, R, S, Y or deletion 57 T E, H, I, N, P or S

TABLE G6 Example substitutions of amino acid residues in SEQ ID NO: 6Kabat numbering Residue Substitution  93 A G, I, N, P or Y  94 R A, K,N, S or T  95 A F, R, T, E, I, L, S, V, H, Q, D, N or deletion  96 Y A,R, I, G, D, S, L, K or deletion  97 F A, Y, W, L, S or R  98 G P  99 N Hor Y 100 S A, T or V 100A F L or M 101 A D 102 Y F, H, N or S

In some embodiments of the antibody or fragment thereof, the CDRL1comprises the amino acid sequence of SEQ ID NO:1, 17 or 18, the CDRL2comprises the amino acid sequence of SEQ ID NO:2, the CDRL3 comprisesthe amino acid sequence of SEQ ID NO:3, 19 or 20, the CDRH1 comprisesthe amino acid sequence of SEQ ID NO:4, the CDRH2 comprises the aminoacid sequence of SEQ ID NO:5 or 21, and the CDRH3 comprises the aminoacid sequence of SEQ ID NO:6 or 22.

Non-limiting examples of a light chain variable region includes an aminoacid sequence selected from the group consisting of SEQ ID NO:141,192-195 and 206-207, or a biological equivalent, e.g., a peptide havingat least 90% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO:141, 192-195 and 206-207.

Non-limiting examples of a heavy chain variable region includes an aminoacid sequence selected from the group consisting of SEQ ID NO:171,188-191 and 205, or a biological equivalent, e.g., a peptide having atleast 90% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO:171, 188-191 and 205.

In some embodiments, the CDRL1 comprises the amino acid sequence of SEQID NO:17, the CDRL2 comprises the amino acid sequence of SEQ ID NO:2,the CDRL3 comprises the amino acid sequence of SEQ ID NO:19, the CDRH1comprises the amino acid sequence of SEQ ID NO:4, the CDRH2 comprisesthe amino acid sequence of SEQ ID NO:21, and the CDRH3 comprises theamino acid sequence of SEQ ID NO:22.

In some embodiments, the antibody or fragment thereof include a lightchain variable region comprising an amino acid sequence of SEQ ID NO:206and a heavy chain variable region comprising an amino acid sequence ofSEQ ID NO:205, or their biological equivalents thereof.

Tables A′-D′ provide the CDR sequences according to Kabat numbering. Itis apparent to the skilled artisan that the substitutions disclosedabove can be readily translated to the CDR according to a differentnumbering scheme.

In some embodiments, an antibody or fragment thereof is provided havingbinding specificity to a wild-type human claudin 18.2 (CLDN18.2)protein. In some embodiments, the antibody or fragment thereof comprisesa light chain variable region comprising light chain complementaritydetermining regions CDRL1, CDRL2, and CDRL3 and a heavy chain variableregion comprising heavy chain complementarity determining regions CDRH1,CDRH2, and CDRH3, and wherein: the CDRL1 comprises the amino acidsequence of SEQ ID NO:210, 304 or 305 or an amino acid sequence havingone, two or three amino acid substitution from SEQ ID NO:210, 304 or305, the CDRL2 comprises the amino acid sequence of SEQ ID NO:227 or anamino acid sequence having one or two amino acid substitution from SEQID NO:227, the CDRL3 comprises the amino acid sequence of SEQ ID NO:3,19 or 20 or an amino acid sequence having one, two or three amino acidsubstitution from SEQ ID NO:3, 19 or 20, the CDRH1 comprises the aminoacid sequence of SEQ ID NO:253 or an amino acid sequence having one, twoor three amino acid substitution from SEQ ID NO: 253, the CDRH2comprises the amino acid sequence of SEQ ID NO:278 or 306 or an aminoacid sequence having one, two or three amino acid substitution from SEQID NO:278 or 306, and the CDRH3 comprises the amino acid sequence of SEQID NO:303 or 307 or an amino acid sequence having one, two or threeamino acid substitution from SEQ ID NO:303 or 307.

In some embodiments, the CDRL1 comprises the amino acid sequence of SEQID NO:210, 304 or 305, the CDRL2 comprises the amino acid sequence ofSEQ ID NO:227, the CDRL3 comprises the amino acid sequence of SEQ IDNO:3, 19 or 20, the CDRH1 comprises the amino acid sequence of SEQ IDNO:253, the CDRH2 comprises the amino acid sequence of SEQ ID NO:278 or306, and the CDRH3 comprises the amino acid sequence of SEQ ID NO:303 or307.

Non-limiting examples of a light chain variable region includes an aminoacid sequence selected from the group consisting of SEQ ID NO:141,192-195 and 206-207, or a biological equivalent, such as a peptidehaving at least 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:141, 192-195 and 206-207.

Non-limiting examples of a heavy chain variable region include an aminoacid sequence selected from the group consisting of SEQ ID NO:171,188-191 and 205, or a biological equivalent, such as a peptide having atleast 90% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO:171, 188-191 and 205.

In some embodiments, the CDRL1 comprises the amino acid sequence of SEQID NO:304, the CDRL2 comprises the amino acid sequence of SEQ ID NO:227,the CDRL3 comprises the amino acid sequence of SEQ ID NO:19, the CDRH1comprises the amino acid sequence of SEQ ID NO:253, the CDRH2 comprisesthe amino acid sequence of SEQ ID NO:306, and the CDRH3 comprises theamino acid sequence of SEQ ID NO:307. A non-limiting example of theantibody or fragment includes a light chain variable region comprisingan amino acid sequence of SEQ ID NO:206 and a heavy chain variableregion comprising an amino acid sequence of SEQ ID NO:205.

Likewise, 72C1B6A3 has been shown to be a good antibody. In anotherembodiment, therefore, provided is an antibody or fragment thereofhaving binding specificity to a wild-type human claudin 18.2 (CLDN18.2)protein, wherein the antibody or fragment thereof comprises a lightchain variable region comprising light chain complementarity determiningregions CDRL1, CDRL2, and CDRL3 and a heavy chain variable regioncomprising heavy chain complementarity determining regions CDRH1, CDRH2,and CDRH3, and wherein: the CDRL1 comprises the amino acid sequence ofQSLLNSGNQKNY (SEQ ID NO:1), QSLLNAGNQKNY (SEQ ID NO:17) or QSLLESGNQKNY(SEQ ID NO:18) or an amino acid sequence having one, two or three aminoacid substitution from SEQ ID NO:1, 17 or 18, the CDRL2 comprises theamino acid sequence of RAS (SEQ ID NO:7) or an amino acid sequencehaving one or two amino acid substitution from SEQ ID NO:7, the CDRL3comprises the amino acid sequence of QNDYIYPYT (SEQ ID NO:8) or an aminoacid sequence having one, two or three amino acid substitution from SEQID NO:8, the CDRH1 comprises the amino acid sequence of GYTFTTYP (SEQ IDNO:9) or an amino acid sequence having one, two or three amino acidsubstitution from SEQ ID NO:9, the CDRH2 comprises the amino acidsequence of FHPYNDDT (SEQ ID NO:10) or an amino acid sequence havingone, two or three amino acid substitution from SEQ ID NO:10, and theCDRH3 comprises the amino acid sequence of ARRAYGYPYAMDY (SEQ ID NO:11)or an amino acid sequence having one, two or three amino acidsubstitution from SEQ ID NO:11.

Likewise, each particular amino acid in a CDR can be substituted withanother amino acid present in a corresponding CDR from a differentantibody. In some embodiments, therefore, the following tables summarizesome feasible substitutions based on sequence alignments.

TABLE H1 Example substitutions of amino acid residues in SEQ ID NO: 1Kabat numbering Residue Substitution 27 Q M 29 L V 30 L F 30B S G 30E QL or R 30F K R 31 N K or S

TABLE H2 Example substitutions of amino acid residues in SEQ ID NO: 7Kabat numbering Residue Substitution 50 R G or W 51 A S 52 S F

TABLE H3 Example substitutions of amino acid residues in SEQ ID NO: 8Kabat numbering Residue Substitution 89 Q H 91 D A, G, N, S or V 92 Y For L 93 I F, Y or S 94 Y F 96 Y C, L, W, or F 97 T A

TABLE H4 Example substitutions of amino acid residues in SEQ ID NO: 9Kabat numbering Residue Substitution 26 G D 27 Y F or L 28 T A or S 29 FI or L 30 T I, N, P or S 31 T K, N, S or G 32 Y F, G or N 33 P A, G, L,I, S or Y

TABLE H5 Example substitutions of amino acid residues in SEQ ID NO: 10Kabat numbering Residue Substitution 51 F I or M 52 H N, I, L, R, S, Tor W   52A P A, G, S, T or Y 53 Y D, G, K, N, S or T 54 N D, G, R, S orT 55 D G, R, S or T 56 D G, R, S, Y or deletion 57 T E, H, I, N, P or S

TABLE H6 Example substitutions of amino acid residues in SEQ ID NO: 11Kabat numbering Residue Substitution 93 A G, I, N, P or Y 94 R A, K, N,S or T 95 R F, A, T, E, I, L, S, V, H, Q, D, N or deletion 96 A Y, R, I,G, D, S, L, K or deletion 97 Y A, F, W, L, S or R 98 G P 99 Y H or N 100P S, A, T or V  100A Y F, L or M 101 A D 102 M Y, F, H, N or S

In some embodiments of the antibody or fragment thereof of 23, the CDRL1comprises the amino acid sequence of SEQ ID NO:1, 17 or 18, the CDRL2comprises the amino acid sequence of SEQ ID NO:7, the CDRL3 comprisesthe amino acid sequence of SEQ ID NO:8, the CDRH1 comprises the aminoacid sequence of SEQ ID NO:9, the CDRH2 comprises the amino acidsequence of SEQ ID NO:10, and the CDRH3 comprises the amino acidsequence of SEQ ID NO:11.

Non-limiting examples of a light chain variable region includes an aminoacid sequence selected from the group consisting of SEQ ID NO:124,185-187 and 203-204, or a biological equivalents, e.g., a peptide havingat least 90% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO:124, 185-187 and 203-204.

Non-limiting examples of a heavy chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NO:153and 181-184, or a biological equivalent, e.g., a peptide having at least90% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:153 and 181-184.

In some embodiments, in the antibody or fragment thereof, the CDRL1comprises the amino acid sequence of SEQ ID NO:17, the CDRL2 comprisesthe amino acid sequence of SEQ ID NO:7, the CDRL3 comprises the aminoacid sequence of SEQ ID NO:8, the CDRH1 comprises the amino acidsequence of SEQ ID NO:9, the CDRH2 comprises the amino acid sequence ofSEQ ID NO:10, and the CDRH3 comprises the amino acid sequence of SEQ IDNO:11.

The antibody or fragment thereof, in some embodiments, includes a lightchain variable region comprising an amino acid sequence of SEQ ID NO:203and a heavy chain variable region comprising an amino acid sequence ofSEQ ID NO:181, or their corresponding biological equivalents.

Tables A′-D′ provide the CDR sequences according to Kabat numbering. Itis apparent to the skilled artisan that the substitutions disclosedabove can be readily translated to the CDR according to a differentnumbering scheme.

In some embodiments, provided is an antibody or fragment thereof havingbinding specificity to a wild-type human claudin 18.2 (CLDN18.2)protein, wherein the antibody or fragment thereof comprises a lightchain variable region comprising light chain complementarity determiningregions CDRL1, CDRL2, and CDRL3 and a heavy chain variable regioncomprising heavy chain complementarity determining regions CDRH1, CDRH2,and CDRH3, and wherein: the CDRL1 comprises the amino acid sequence ofSEQ ID NO:210, 304 or 305 or an amino acid sequence having one, two orthree amino acid substitution from SEQ ID NO:210, 304 or 305, the CDRL2comprises the amino acid sequence of SEQ ID NO:229 or an amino acidsequence having one or two amino acid substitution from SEQ ID NO:229,the CDRL3 comprises the amino acid sequence of SEQ ID NO:8 or an aminoacid sequence having one, two or three amino acid substitution from SEQID NO:8, the CDRH1 comprises the amino acid sequence of SEQ ID NO:242 oran amino acid sequence having one, two or three amino acid substitutionfrom SEQ ID NO:242, the CDRH2 comprises the amino acid sequence of SEQID NO:263 or an amino acid sequence having one, two or three amino acidsubstitution from SEQ ID NO:263, and the CDRH3 comprises the amino acidsequence of SEQ ID NO:289 or an amino acid sequence having one, two orthree amino acid substitution from SEQ ID NO:289.

In some embodiments, the CDRL1 comprises the amino acid sequence of SEQID NO:210, 304 or 305, the CDRL2 comprises the amino acid sequence ofSEQ ID NO:229, the CDRL3 comprises the amino acid sequence of SEQ IDNO:8, the CDRH1 comprises the amino acid sequence of SEQ ID NO:242, theCDRH2 comprises the amino acid sequence of SEQ ID NO:263, and the CDRH3comprises the amino acid sequence of SEQ ID NO:289.

Non-limiting examples of a light chain variable region include an aminoacid sequence selected from the group consisting of SEQ ID NO:124,185-187 and 203-204, or a biological equivalent, such as a peptidehaving at least 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:124, 185-187 and 203-204.

Non-limiting examples of a heavy chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NO:153and 181-184, or a biological equivalent, such as a peptide having atleast 90% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO:153 and 181-184.

In some embodiments, the CDRL1 comprises the amino acid sequence of SEQID NO:304, the CDRL2 comprises the amino acid sequence of SEQ ID NO:229,the CDRL3 comprises the amino acid sequence of SEQ ID NO:8, the CDRH1comprises the amino acid sequence of SEQ ID NO:242, the CDRH2 comprisesthe amino acid sequence of SEQ ID NO:263, and the CDRH3 comprises theamino acid sequence of SEQ ID NO:289. A non-limiting example antibody orfragment thereof includes a light chain variable region comprising anamino acid sequence of SEQ ID NO:203 and a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:181.

Also, 4F11E2 has been shown to be a good antibody. In anotherembodiment, therefore, provided is an antibody or fragment thereofhaving binding specificity to a wild-type human claudin 18.2 (CLDN18.2)protein, wherein the antibody or fragment thereof comprises a lightchain variable region comprising light chain complementarity determiningregions CDRL1, CDRL2, and CDRL3 and a heavy chain variable regioncomprising heavy chain complementarity determining regions CDRH1, CDRH2,and CDRH3, and wherein: the CDRL1 comprises the amino acid sequence ofQSLLNSGNRKNY (SEQ ID NO:12), QSLLESGNRKNY (SEQ ID NO:23) or QSLLNAGNRKNY(SEQ ID NO:24) or an amino acid sequence having one, two or three aminoacid substitution from SEQ ID NO:12, 23 or 24, the CDRL2 comprises theamino acid sequence of WAS (SEQ ID NO:2) or an amino acid sequencehaving one or two amino acid substitution from SEQ ID NO:2, the CDRL3comprises the amino acid sequence of QNAYSYPFT (SEQ ID NO:13) or anamino acid sequence having one, two or three amino acid substitutionfrom SEQ ID NO:13, the CDRH1 comprises the amino acid sequence ofGFTFSTFG (SEQ ID NO:14) or an amino acid sequence having one, two orthree amino acid substitution from SEQ ID NO:14, the CDRH2 comprises theamino acid sequence of ITSGNSPI (SEQ ID NO:15), ITSGQSPI (SEQ ID NO:25),or ITSGESPI (SEQ ID NO:26) or an amino acid sequence having one, two orthree amino acid substitution from SEQ ID NO:15, 25 or 26, and the CDRH3comprises the amino acid sequence of ARSSYYGNSMDY (SEQ ID NO:16),ARSSYYGQSMDY (SEQ ID NO:27), ARSSYYGESMDY (SEQ ID NO:28), orARSSYYGNAMDY (SEQ ID NO:29) or an amino acid sequence having one, two orthree amino acid substitution from SEQ ID NO:16, 27, 28 or 29.

Likewise, each particular amino acid in a CDR can be substituted withanother amino acid present in a corresponding CDR from a differentantibody. In some embodiments, therefore, the following tables summarizesome feasible substitutions based on sequence alignments.

TABLE I1 Example substitutions of amino acid residues in SEQ ID NO: 12Kabat numbering Residue Substitution 27 Q M 29 L V 30 L F  30B S G  30ER L or Q  30F K R 31 N K or S

TABLE I2 Example substitutions of amino acid residues in SEQ ID NO: 2Kabat numbering Residue Substitution 50 W G or R 51 A S 52 S F

TABLE I3 Example substitutions of amino acid residues in SEQ ID NO: 13Kabat numbering Residue Substitution 89 Q H 91 A G, D, N, S or V 92 Y For L 93 S F, I or Y 94 Y F 96 F C, L, W, or Y 97 T A

TABLE I4 Example substitutions of amino acid residues in SEQ ID NO: 14Kabat numbering Residue Substitution 26 G D 27 F Y or L 28 T A or S 29 FI or L 30 S I, N, P or T 31 T K, N, S or G 32 F Y, G or N 33 G A, I, L,P, S or Y

TABLE I5 Example substitutions of amino acid residues in SEQ ID NO: 15Kabat numbering Residue Substitution 51 I F or M 52 T H, I, L, R, S, Nor W  52A S A, G, P, T or Y 53 G D, Y, K, N, S or T 54 N D, G, R, S or T55 S G, R, D or T 56 P G, D, R, S, Y or deletion 57 I E, H, T, N, P or S

TABLE I6 Example substitutions of amino acid residues in SEQ ID NO: 16Kabat numbering Residue Substitution 93 A G, I, N, P or Y 94 R A, K, N,S or T 95 S F, R, T, E, I, L, A, V, H, Q, D, N or deletion 96 S A, R, I,G, D, Y, L, K or deletion 97 Y F, A, Y, W, L, S or R 98 Y G or P 99 N Hor Y 100 S A, T or V   100A M L or F 101 D A 102 Y F, H, N or S

In some embodiments, in the antibody or fragment thereof, the CDRL1comprises the amino acid sequence of SEQ ID NO:12, 23 or 24, the CDRL2comprises the amino acid sequence of SEQ ID NO:2, the CDRL3 comprisesthe amino acid sequence of SEQ ID NO:13, the CDRH1 comprises the aminoacid sequence of SEQ ID NO:14, the CDRH2 comprises the amino acidsequence of SEQ ID NO:15, 25 or 26, and the CDRH3 comprises the aminoacid sequence of SEQ ID NO:16, 27, 28 or 29.

Non-limiting examples of a light chain variable region includes an aminoacid sequence selected from the group consisting of SEQ ID NO:129,178-180 and 201-202, or their biological equivalents, e.g., a peptidehaving at least 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:129, 178-180 and 201-202.

Non-limiting examples of a heavy chain variable region includes an aminoacid sequence selected from the group consisting of SEQ ID NO:159,175-177 and 196-200, or their biological equivalents, e.g., a peptidehaving at least 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:159, 175-177 and 196-200.

In some embodiments, the CDRL1 comprises the amino acid sequence of SEQID NO:24, the CDRL2 comprises the amino acid sequence of SEQ ID NO:2,the CDRL3 comprises the amino acid sequence of SEQ ID NO:13, the CDRH1comprises the amino acid sequence of SEQ ID NO:14, the CDRH2 comprisesthe amino acid sequence of SEQ ID NO:26, and the CDRH3 comprises theamino acid sequence of SEQ ID NO:16.

In some embodiments, the antibody or fragment thereof includes a lightchain variable region comprising an amino acid sequence of SEQ ID NO:202and a heavy chain variable region comprising an amino acid sequence ofSEQ ID NO:197, or their corresponding biological equivalents.

Tables A′-D′ provide the CDR sequences according to Kabat numbering. Itis apparent to the skilled artisan that the substitutions disclosedabove can be readily translated to the CDR according to a differentnumbering scheme.

Some embodiments of the present disclosure provide an antibody orfragment thereof having binding specificity to a wild-type human claudin18.2 (CLDN18.2) protein, wherein the antibody or fragment thereofcomprises a light chain variable region comprising light chaincomplementarity determining regions CDRL1, CDRL2, and CDRL3 and a heavychain variable region comprising heavy chain complementarity determiningregions CDRH1, CDRH2, and CDRH3, and wherein: the CDRL1 comprises theamino acid sequence of SEQ ID NO:216, 308 or 309 or an amino acidsequence having one, two or three amino acid substitution from SEQ IDNO:216, 308 or 309, the CDRL2 comprises the amino acid sequence of SEQID NO:227 or an amino acid sequence having one or two amino acidsubstitution from SEQ ID NO:227, the CDRL3 comprises the amino acidsequence of SEQ ID NO:13 or an amino acid sequence having one, two orthree amino acid substitution from SEQ ID NO:13, the CDRH1 comprises theamino acid sequence of SEQ ID NO:246 or an amino acid sequence havingone, two or three amino acid substitution from SEQ ID NO:246, the CDRH2comprises the amino acid sequence of SEQ ID NO:268, 310 or 311 or anamino acid sequence having one, two or three amino acid substitutionfrom SEQ ID NO:268, 310 or 311, and the CDRH3 comprises the amino acidsequence of SEQ ID NO:294, 312, 313 or 314, or an amino acid sequencehaving one, two or three amino acid substitution from SEQ ID NO:294,312, 313 or 314.

In some embodiments, the CDRL1 comprises the amino acid sequence of SEQID NO:216, 308 or 309, the CDRL2 comprises the amino acid sequence ofSEQ ID NO:227, the CDRL3 comprises the amino acid sequence of SEQ IDNO:13, the CDRH1 comprises the amino acid sequence of SEQ ID NO:246, theCDRH2 comprises the amino acid sequence of SEQ ID NO:268, 310 or 311,and the CDRH3 comprises the amino acid sequence of SEQ ID NO:294, 312,313 or 314.

Non-limiting examples of a light chain variable region include an aminoacid sequence selected from the group consisting of SEQ ID NO:129,178-180 and 201-202, or a biological equivalent, such as a peptidehaving at least 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:129, 178-180 and 201-202.

Non-limiting examples of a heavy chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NO:159,175-177 and 196-200, or a biological equivalents, such as a peptidehaving at least 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:159, 175-177 and 196-200.

In some embodiments, the CDRL1 comprises the amino acid sequence of SEQID NO:309, the CDRL2 comprises the amino acid sequence of SEQ ID NO:227,the CDRL3 comprises the amino acid sequence of SEQ ID NO:13, the CDRH1comprises the amino acid sequence of SEQ ID NO:246, the CDRH2 comprisesthe amino acid sequence of SEQ ID NO:311, and the CDRH3 comprises theamino acid sequence of SEQ ID NO:294. A non-limiting example of theantibody or fragment includes a light chain variable region comprisingan amino acid sequence of SEQ ID NO:202 and a heavy chain variableregion comprising an amino acid sequence of SEQ ID NO:197.

In some embodiments, the antibodies are humanized antibodies. Humanizedantibodies, as shown in Example 9, can include one or more backmutations to the mouse counterpart. Examples of such back mutations areshown in Table 3. In some embodiments, the antibody or fragment caninclude one, two, three, four, five or more of the back mutations.

In some embodiments, the anti-claudin 18.2 antibody of the presentdisclosure includes a VL of any one of SEQ ID NO: 117-144, 178-180,185-187, 192-195, 201-202, 203-204 or 206-207, and a VH of any one ofSEQ ID NO: 145-174, 175-177, 181-184, 188-191, 196-200, or 205 or theirrespective biological equivalents. A biological equivalent of a VH or VLis a sequence that includes the designated amino acids while having anoverall 80%, 85%, 90%, 95%, 98% or 99% sequence identity. A biologicalequivalent of SEQ ID NO:145, therefore, can be a VH that has an overall80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO:145 butretains the CDRs, and optionally retains one or more, or all of theback-mutations.

It will also be understood by one of ordinary skill in the art thatantibodies as disclosed herein may be modified such that they vary inamino acid sequence from the naturally occurring binding polypeptidefrom which they were derived. For example, a polypeptide or amino acidsequence derived from a designated protein may be similar, e.g., have acertain percent identity to the starting sequence, e.g., it may be 60%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the startingsequence.

In certain embodiments, the antibody comprises an amino acid sequence orone or more moieties not normally associated with an antibody. Exemplarymodifications are described in more detail below. For example, anantibody of the disclosure may comprise a flexible linker sequence, ormay be modified to add a functional moiety (e.g., PEG, a drug, a toxin,or a label).

Antibodies, variants, or derivatives thereof of the disclosure includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the antibody such that covalent attachment does notprevent the antibody from binding to the epitope. For example, but notby way of limitation, the antibodies can be modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the antibodies may contain one or more non-classicalamino acids.

In some embodiments, the antibodies may be conjugated to therapeuticagents, prodrugs, peptides, proteins, enzymes, viruses, lipids,biological response modifiers, pharmaceutical agents, or PEG.

The antibodies may be conjugated or fused to a therapeutic agent, whichmay include detectable labels such as radioactive labels, animmunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactivetherapeutic or diagnostic agent, a cytotoxic agent, which may be a drugor a toxin, an ultrasound enhancing agent, a non-radioactive label, acombination thereof and other such agents known in the art.

The antibodies can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantigen-binding polypeptide is then determined by detecting the presenceof luminescence that arises during the course of a chemical reaction.Examples of particularly useful chemiluminescent labeling compounds areluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt and oxalate ester.

The antibodies can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA). Techniques for conjugatingvarious moieties to an antibody are well known, see, e.g., Arnon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies ForDrug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.,(eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. (52:119-58 (1982)).

Bi-Functional Molecules

As a tumor antigen targeting molecule, an antibody or antigen-bindingfragment specific to claudin 18.2 can be combined with a second fragmentspecific to an immune cell to generate a bispecific antibody.

In some embodiments, the immune cell is selected from the groupconsisting of a T cell, a B cell, a monocyte, a macrophage, aneutrophil, a dendritic cell, a phagocyte, a natural killer cell, aneosinophil, a basophil, and a mast cell.

In some embodiment, the second specificity is to CD3, CD47, PD1, PD-L1,LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2,CMET, 4-1BB, OX40, SIRPA CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM,CD27, VEGFR, or VEGF.

Different format of bispecific antibodies are also provided. In someembodiments, each of the anti-claudin 18.2 fragment and the secondfragment each is independently selected from a Fab fragment, asingle-chain variable fragment (scFv), or a single-domain antibody. Insome embodiments, the bispecific antibody further includes a Fcfragment.

Bifunctional molecules that include not just antibody or antigen bindingfragment are also provided. As a tumor antigen targeting molecule, anantibody or antigen-binding fragment specific to claudin 18.2, such asthose described here, can be combined with an immune cytokine or ligandoptionally through a peptide linker. The linked immune cytokines orligands include, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L,4-1BBL, LIGHT and GITRL. Such bi-functional molecules can combine theimmune checkpoint blocking effect with tumor site local immunemodulation.

Polynucleotides Encoding the Antibodies and Methods of Preparing theAntibodies

The present disclosure also provides isolated polynucleotides or nucleicacid molecules encoding the antibodies, variants or derivatives thereofof the disclosure. The polynucleotides of the present disclosure mayencode the entire heavy and light chain variable regions of theantigen-binding polypeptides, variants or derivatives thereof on thesame polynucleotide molecule or on separate polynucleotide molecules.Additionally, the polynucleotides of the present disclosure may encodeportions of the heavy and light chain variable regions of theantigen-binding polypeptides, variants or derivatives thereof on thesame polynucleotide molecule or on separate polynucleotide molecules.

Methods of making antibodies are well known in the art and describedherein. In certain embodiments, both the variable and constant regionsof the antigen-binding polypeptides of the present disclosure are fullyhuman Fully human antibodies can be made using techniques described inthe art and as described herein. For example, fully human antibodiesagainst a specific antigen can be prepared by administering the antigento a transgenic animal which has been modified to produce suchantibodies in response to antigenic challenge, but whose endogenous locihave been disabled. Exemplary techniques that can be used to make suchantibodies are described in U.S. Pat. Nos. 6,150,584; 6,458,592;6,420,140 which are incorporated by reference in their entireties.

Treatment Methods

As described herein, the antibodies, variants or derivatives of thepresent disclosure may be used in certain treatment and diagnosticmethods.

The present disclosure is further directed to antibody-based therapieswhich involve administering the antibodies of the disclosure to apatient such as an animal, a mammal, and a human for treating one ormore of the disorders or conditions described herein. Therapeuticcompounds of the disclosure include, but are not limited to, antibodiesof the disclosure (including variants and derivatives thereof asdescribed herein) and nucleic acids or polynucleotides encodingantibodies of the disclosure (including variants and derivatives thereofas described herein).

The antibodies of the disclosure can also be used to treat or inhibitcancer. As provided above, claudin 18.2 can be overexpressed in tumorcells, in particular gastric, pancreatic, esophageal, ovarian, and lungtumors. Inhibition of claudin 18.2 has been shown to be useful fortreating the tumors.

Accordingly, in some embodiments, provided are methods for treating acancer in a patient in need thereof. The method, in one embodiment,entails administering to the patient an effective amount of an antibodyof the present disclosure. In some embodiments, at least one of thecancer cells (e.g., stromal cells) in the patient over-express claudin18.2.

Cellular therapies, such as chimeric antigen receptor (CAR) T-celltherapies, are also provided in the present disclosure. A suitable cellcan be used, that is put in contact with an anti-claudin 18.2 antibodyof the present disclosure (or alternatively engineered to express ananti-claudin 18.2 antibody of the present disclosure). Upon such contactor engineering, the cell can then be introduced to a cancer patient inneed of a treatment. The cancer patient may have a cancer of any of thetypes as disclosed herein. The cell (e.g., T cell) can be, for instance,a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or thecombination thereof, without limitation.

In some embodiments, the cell was isolated from the cancer patient him-or her-self. In some embodiments, the cell was provided by a donor orfrom a cell bank. When the cell is isolated from the cancer patient,undesired immune reactions can be minimized.

Non-limiting examples of cancers include bladder cancer, breast cancer,colorectal cancer, endometrial cancer, esophageal cancer, head and neckcancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma,melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. Insome embodiments, the cancer is one or more of gastric, pancreatic,esophageal, ovarian, and lung cancers.

Additional diseases or conditions associated with increased cellsurvival, that may be treated, prevented, diagnosed and/or prognosedwith the antibodies or variants, or derivatives thereof of thedisclosure include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyo sarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma.

A specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the particular antibodies,variant or derivative thereof used, the patient's age, body weight,general health, sex, and diet, and the time of administration, rate ofexcretion, drug combination, and the severity of the particular diseasebeing treated. Judgment of such factors by medical caregivers is withinthe ordinary skill in the art. The amount will also depend on theindividual patient to be treated, the route of administration, the typeof formulation, the characteristics of the compound used, the severityof the disease, and the desired effect. The amount used can bedetermined by pharmacological and pharmacokinetic principles well knownin the art.

Methods of administration of the antibodies, variants or include but arenot limited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The antigen-bindingpolypeptides or compositions may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Thus, pharmaceutical compositions containingthe antigen-binding polypeptides of the disclosure may be administeredorally, rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, drops ortransdermal patch), bucally, or as an oral or nasal spray.

The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intra-articular injection and infusion.

Administration can be systemic or local. In addition, it may bedesirable to introduce the antibodies of the disclosure into the centralnervous system by any suitable route, including intraventricular andintrathecal injection; intraventricular injection may be facilitated byan intraventricular catheter, for example, attached to a reservoir, suchas an Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

It may be desirable to administer the antigen-binding polypeptides orcompositions of the disclosure locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, topical application, e.g., inconjunction, with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering a protein, including an antibody, of the disclosure,care must be taken to use materials to which the protein does notabsorb.

The amount of the antibodies of the disclosure which will be effectivein the treatment, inhibition and prevention of an inflammatory, immuneor malignant disease, disorder or condition can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease, disorder orcondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

As a general proposition, the dosage administered to a patient of theantigen-binding polypeptides of the present disclosure is typically 0.1mg/kg to 100 mg/kg of the patient's body weight, between 0.1 mg/kg and20 mg/kg of the patient's body weight, or 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of thedisclosure may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

In an additional embodiment, the compositions of the disclosure areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the disclosure include, but arenot limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13,IL-15, anti-CD40, CD40L, and TNF-α.

In additional embodiments, the compositions of the disclosure areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Combination Therapies and Antibody-Drug Conjugates

Combination therapies are also provided, which includes the use of oneor more of the anti-claudin 18.2 antibodies or fragments thereof of thepresent disclosure along with a second anticancer (chemotherapeutic)agent. In some embodiment, the chemotherapeutic agent is conjugated tothe antibody or fragment.

Chemotherapeutic agents may be categorized by their mechanism of actioninto, for example, the following groups:

anti-metabolites/anti-cancer agents such as pyrimidine analogsfloxuridine, capecitabine, and cytarabine;

purine analogs, folate antagonists, and related inhibitors;

antiproliferative/antimitotic agents including natural products such asvinca alkaloid (vinblastine, vincristine) and microtubule such as taxane(paclitaxel, docetaxel), vinblastin, nocodazole, epothilones,vinorelbine (NAVELBINE®), and epipodophyllotoxins (etoposide,teniposide);

DNA damaging agents such as actinomycin, amsacrine, busulfan,carboplatin, chlorambucil, cisplatin, cyclophosphamide (CYTOXAN®),dactinomycin, daunorubicin, doxorubicin, epirubicin, iphosphamide,melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea,procarbazine, taxol, taxotere, teniposide, etoposide, andtriethylenethiophosphoramide;

antibiotics such as dactinomycin, daunorubicin, doxorubicin, idarubicin,anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin), andmitomycin;

enzymes such as L-asparaginase which systemically metabolizesL-asparagine and deprives cells which do not have the capacity tosynthesize their own asparagine;

antiplatelet agents;

antiproliferative/antimitotic alkylating agents such as nitrogenmustards cyclophosphamide and analogs (melphalan, chlorambucil,hexamethylmelamine, and thiotepa), alkyl nitrosoureas (carmustine) andanalogs, streptozocin, and triazenes (dacarbazine);

antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate);

platinum coordination complexes (cisplatin, oxiloplatinim, andcarboplatin), procarbazine, hydroxyurea, mitotane, andaminoglutethimide;

hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,and nilutamide), and aromatase inhibitors (letrozole and anastrozole);

anticoagulants such as heparin, synthetic heparin salts, and otherinhibitors of thrombin;

fibrinolytic agents such as tissue plasminogen activator, streptokinase,urokinase, aspirin, dipyridamole, ticlopidine, and clopidogrel;

antimigratory agents;

antisecretory agents (breveldin);

immunosuppressives tacrolimus, sirolimus, azathioprine, andmycophenolate;

compounds (TNP-470, genistein) and growth factor inhibitors (vascularendothelial growth factor inhibitors and fibroblast growth factorinhibitors);

angiotensin receptor blockers, nitric oxide donors;

anti-sense oligonucleotides;

antibodies such as trastuzumab and rituximab;

cell cycle inhibitors and differentiation inducers such as tretinoin;

inhibitors, topoisomerase inhibitors (doxorubicin, daunorubicin,dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan,mitoxantrone, topotecan, and irinotecan), and corticosteroids(cortisone, dexamethasone, hydrocortisone, methylprednisolone,prednisone, and prednisolone);

growth factor signal transduction kinase inhibitors;

dysfunction inducers;

toxins such as Cholera toxin, ricin, Pseudomonas exotoxin, Bordetellapertussis adenylate cyclase toxin, diphtheria toxin, and caspaseactivators;

and chromatin.

Further examples of chemotherapeutic agents include:

alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®);

alkyl sulfonates such as busulfan, improsulfan, and piposulfan;

aziridines such as benzodopa, carboquone, meturedopa, and uredopa;

emylerumines and memylamelamines including alfretamine,triemylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimemylolomelamine;

acetogenins, especially bullatacin and bullatacinone;

a camptothecin, including synthetic analog topotecan;

bryostatin;

callystatin;

CC-1065, including its adozelesin, carzelesin, and bizelesin syntheticanalogs;

cryptophycins, particularly cryptophycin 1 and cryptophycin 8;

dolastatin;

duocarmycin, including the synthetic analogs KW-2189 and CBI-TMI;

eleutherobin;

pancratistatin;

a sarcodictyin;

spongistatin;

nitrogen mustards such as chlorambucil, chlornaphazine,cyclophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;

nitrosoureas such as carmustine, chlorozotocin, foremustine, lomustine,nimustine, and ranimustine;

antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammaII and calicheamicin phiI1), dynemicinincluding dynemicin A, bisphosphonates such as clodronate, anesperamicin, neocarzinostatin chromophore and related chromoproteinenediyne antibiotic chromomophores, aclacinomycins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, carabicin,carrninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin,detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin;

anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);

folic acid analogs such as demopterin, methotrexate, pteropterin, andtrimetrexate;

purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, andthioguanine;

pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, andfloxuridine;

androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone;

anti-adrenals such as aminoglutethimide, mitotane, and trilostane;

folic acid replinishers such as frolinic acid;

trichothecenes, especially T-2 toxin, verracurin A, roridin A, andanguidine;

taxoids such as paclitaxel (TAXOL®) and docetaxel (TAXOTERE®);

platinum analogs such as cisplatin and carboplatin;

aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; leucovorin; lonidamine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;losoxantrone; fluoropyrimidine; folinic acid; podophyllinic acid;2-ethylhydrazide; procarbazine; polysaccharide-K (PSK); razoxane;rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-tricUorotriemylamine; urethane; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiopeta; chlorambucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitroxantrone;vancristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO);retinoids such as retinoic acid; capecitabine; FOLFIRI (fluorouracil,leucovorin, and irinotecan);

and pharmaceutically acceptable salts, acids, or derivatives of any ofthe above.

Also included in the definition of “chemotherapeutic agent” areanti-hormonal agents such as anti-estrogens and selective estrogenreceptor modulators (SERMs), inhibitors of the enzyme aromatase,anti-androgens, and pharmaceutically acceptable salts, acids orderivatives of any of the above that act to regulate or inhibit hormoneaction on tumors.

Examples of anti-estrogens and SERMs include, for example, tamoxifen(including NOLVADEX™), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene(FARESTON®).

Inhibitors of the enzyme aromatase regulate estrogen production in theadrenal glands. Examples include 4(5)-imidazoles, aminoglutethimide,megestrol acetate (MEGACE®), exemestane, formestane, fadrozole, vorozole(RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®).

Examples of anti-androgens include flutamide, nilutamide, bicalutamide,leuprohde, and goserelin.

Examples of chemotherapeutic agents also include anti-angiogenic agentsincluding, but are not limited to, retinoid acid and derivativesthereof, 2-methoxyestradiol, ANGIOSTATIN®, ENDOSTATIN®, suramin,squalamine, tissue inhibitor of metalloproteinase-1, tissue inhibitor ofmetalloproteinase-2, plasminogen activator inhibitor-1, plasminogenactivator inbibitor-2, cartilage-derived inhibitor, paclitaxel(nab-paclitaxel), platelet factor 4, protamine sulphate (clupeine),sulphated chitin derivatives (prepared from queen crab shells),sulphated polysaccharide peptidoglycan complex (sp-pg), staurosporine,modulators of matrix metabolism including proline analogs((1-azetidine-2-carboxylic acid (LACA)), cishydroxyproline,d,I-3,4-dehydroproline, thiaproline, α,α′-dipyridyl,beta-aminopropionitrile fumarate,4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone, methotrexate, mitoxantrone,heparin, interferons, 2 macroglobulin-serum, chicken inhibitor ofmetalloproteinase-3 (ChIMP-3), chymostatin, beta-cyclodextrintetradecasulfate, eponemycin, fumagillin, gold sodium thiomalate,d-penicillamine, beta-1-anticollagenase-serum, alpha-2-antiplasmin,bisantrene, lobenzarit disodium, n-2-carboxyphenyl-4-chloroanthronilicacid disodium or “CCA”, thalidomide, angiostatic steroid, carboxyaminoimidazole, and metalloproteinase inhibitors such as BB-94. Otheranti-angiogenesis agents include antibodies, preferably monoclonalantibodies against these angiogenic growth factors: beta-FGF, alpha-FGF,FGF-5, VEGF isoforms, VEGF-C, HGF/SF, and Ang-1/Ang-2.

Examples of chemotherapeutic agents also include anti-fibrotic agentsincluding, but are not limited to, the compounds such asbeta-aminoproprionitrile (BAPN), as well as the compounds disclosed inU.S. Pat. No. 4,965,288 (Palfreyman, et al.) relating to inhibitors oflysyl oxidase and their use in the treatment of diseases and conditionsassociated with the abnormal deposition of collagen and U.S. Pat. No.4,997,854 (Kagan et al.) relating to compounds which inhibit LOX for thetreatment of various pathological fibrotic states, which are hereinincorporated by reference. Further exemplary inhibitors are described inU.S. Pat. No. 4,943,593 (Palfreyman et al.) relating to compounds suchas 2-isobutyl-3-fluoro-, chloro-, or bromo-allylamine, U.S. Pat. No.5,021,456 (Palfreyman et al.), U.S. Pat. No. 5,059,714 (Palfreyman etal.), U.S. Pat. No. 5,120,764 (Mccarthy et al.), U.S. Pat. No. 5,182,297(Palfreyman et al.), U.S. Pat. No. 5,252,608 (Palfreyman et al.)relating to 2-(1-naphthyloxymemyl)-3-fluoroallylamine, and U.S. Pub.No.: 2004/0248871 (Farjanel et al.), which are herein incorporated byreference.

Exemplary anti-fibrotic agents also include the primary amines reactingwith the carbonyl group of the active site of the lysyl oxidases, andmore particularly those which produce, after binding with the carbonyl,a product stabilized by resonance, such as the following primary amines:emylenemamine, hydrazine, phenylhydrazine, and their derivatives;semicarbazide and urea derivatives; aminonitriles such as BAPN or2-nitroethylamine; unsaturated or saturated haloamines such as2-bromo-ethylamine, 2-chloroethylamine, 2-trifluoroethylamine,3-bromopropylamine, and p-halobenzylamines; and selenohomocysteinelactone.

Other anti-fibrotic agents are copper chelating agents penetrating ornot penetrating the cells. Exemplary compounds include indirectinhibitors which block the aldehyde derivatives originating from theoxidative deamination of the lysyl and hydroxylysyl residues by thelysyl oxidases. Examples include the thiolamines, particularlyD-penicillamine, and its analogs such as2-amino-5-mercapto-5-methylhexanoic acid,D-2-amino-3-methyl-3-((2-acetamidoethyl)dithio)butanoic acid,p-2-amino-3-methyl-3-((2-aminoethyl)dithio)butanoic acid,sodium-4-((p-1-dimethyl-2-amino-2-carboxyethyl)dithio)butane sulphurate,2-acetamidoethyl-2-acetamidoethanethiol sulphanate, andsodium-4-mercaptobutanesulphinate trihydrate.

Examples of chemotherapeutic agents also include immunotherapeuticagents including and are not limited to therapeutic antibodies suitablefor treating patients. Some examples of therapeutic antibodies includesimtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab,altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab,bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab,catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab,conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab,detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab,ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab,figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab,girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab,indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab,labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab,mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab,moxetumomab, narnatumab, naptumomab, necitumumab, nimotuzumab,nofetumomab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab,oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab,pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab,rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab,siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab,teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab,ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49, and3F8. Rituximab can be used for treating indolent B-cell cancers,including marginal-zone lymphoma, WM, CLL and small lymphocyticlymphoma. A combination of Rituximab and chemotherapy agents isespecially effective.

The exemplified therapeutic antibodies may be further labeled orcombined with a radioisotope particle such as indium-111, yttrium-90, oriodine-131.

In a one embodiment, the additional therapeutic agent is a nitrogenmustard alkylating agent. Nonlimiting examples of nitrogen mustardalkylating agents include chlorambucil.

In one embodiment, the compounds and compositions described herein maybe used or combined with one or more additional therapeutic agents. Theone or more therapeutic agents include, but are not limited to, aninhibitor of Abl, activated CDC kinase (ACK), adenosine A2B receptor(A2B), apoptosis signal-regulating kinase (ASK), Auroa kinase, Bruton'styrosine kinase (BTK), BET-bromodomain (BRD) such as BRD4, c-Kit, c-Met,CDK-activating kinase (CAK), calmodulin-dependent protein kinase (CaMK),cyclin-dependent kinase (CDK), casein kinase (CK), discoidin domainreceptor (DDR), epidermal growth factor receptors (EGFR), focal adhesionkinase (FAK), Flt-3, FYN, glycogen synthase kinase (GSK), HCK, histonedeacetylase (HDAC), IKK such as IKKβε, isocitrate dehydrogenase (IDH)such as IDH1, Janus kinase (JAK), KDR, lymphocyte-specific proteintyrosine kinase (LCK), lysyl oxidase protein, lysyl oxidase-like protein(LOXL), LYN, matrix metalloprotease (MMP), MEK, mitogen-activatedprotein kinase (MAPK), NEK9, NPM-ALK, p38 kinase, platelet-derivedgrowth factor (PDGF), phosphorylase kinase (PK), polo-like kinase (PLK),phosphatidylinositol 3-kinase (PI3K), protein kinase (PK) such asprotein kinase A, B, and/or C, PYK, spleen tyrosine kinase (SYK),serine/threonine kinase TPL2, serine/threonine kinase STK, signaltransduction and transcription (STAT), SRC, serine/threonine-proteinkinase (TBK) such as TBK1, TIE, tyrosine kinase (TK), vascularendothelial growth factor receptor (VEGFR), YES, or any combinationthereof.

ASK inhibitors include ASK1 inhibitors. Examples of ASK1 inhibitorsinclude, but are not limited to, those described in WO 2011/008709(Gilead Sciences) and WO 2013/112741 (Gilead Sciences).

Examples of BTK inhibitors include, but are not limited to, ibrutinib,HM71224, ONO-4059, and CC-292.

DDR inhibitors include inhibitors of DDR1 and/or DDR2. Examples of DDRinhibitors include, but are not limited to, those disclosed in WO2014/047624 (Gilead Sciences), US 2009/0142345 (Takeda Pharmaceutical),US 2011/0287011 (Oncomed Pharmaceuticals), WO 2013/027802 (ChugaiPharmaceutical), and WO 2013/034933 (Imperial Innovations).

Examples of HDAC inhibitors include, but are not limited to, pracinostatand panobinostat.

JAK inhibitors inhibit JAK1, JAK2, and/or JAK3. Examples of JAKinhibitors include, but are not limited to, filgotinib, ruxolitinib,fedratinib, tofacitinib, baricitinib, lestaurtinib, pacritinib, XL019,AZD1480, INCB039110, LY2784544, BMS911543, and NS018.

LOXL inhibitors include inhibitors of LOXL1, LOXL2, LOXL3, LOXL4, and/orLOXL5. Examples of LOXL inhibitors include, but are not limited to, theantibodies described in WO 2009/017833 (Arresto Biosciences).

Examples of LOXL2 inhibitors include, but are not limited to, theantibodies described in WO 2009/017833 (Arresto Biosciences), WO2009/035791 (Arresto Biosciences), and WO 2011/097513 (GileadBiologics).

MMP inhibitors include inhibitors of MMP1 through 10. Examples of MMP9inhibitors include, but are not limited to, marimastat (BB-2516),cipemastat (Ro 32-3555), and those described in WO 2012/027721 (GileadBiologics).

PI3K inhibitors include inhibitors of PI3Kγ, PI3Kδ, PI3Kβ, PI3Kα, and/orpan-PI3K. Examples of PI3K inhibitors include, but are not limited to,wortmannin, BKM120, CH5132799, XL756, and GDC-0980.

Examples of PI3Kγ inhibitors include, but are not limited to, ZSTK474,AS252424, LY294002, and TG100115.

Examples of PI3Kδ inhibitors include, but are not limited to, PI3K II,TGR-1202, AMG-319, GSK2269557, X-339, X-414, RP5090, KAR4141, XL499,OXY111A, IPI-145, IPI-443, and the compounds described in WO 2005/113556(ICOS), WO 2013/052699 (Gilead Calistoga), WO 2013/116562 (GileadCalistoga), WO 2014/100765 (Gilead Calistoga), WO 2014/100767 (GileadCalistoga), and WO 2014/201409 (Gilead Sciences).

Examples of PI3Kβ inhibitors include, but are not limited to,GSK2636771, BAY 10824391, and TGX221.

Examples of PI3Kα inhibitors include, but are not limited to,buparlisib, BAY 80-6946, BYL719, PX-866, RG7604, MLN1117, WX-037,AEZA-129, and PA799.

Examples of pan-PI3K inhibitors include, but are not limited to,LY294002, BEZ235, XL147 (SAR245408), and GDC-0941.

Examples of SYK inhibitors include, but are not limited to, tamatinib(R406), fostamatinib (R788), PRT062607, BAY-61-3606, NVP-QAB 205 AA,R112, R343, and those described in U.S. Pat. No. 8,450,321 (GileadConn.).

TKIs may target epidermal growth factor receptors (EGFRs) and receptorsfor fibroblast growth factor (FGF), platelet-derived growth factor(PDGF), and vascular endothelial growth factor (VEGF). Examples of TKIsthat target EGFR include, but are not limited to, gefitinib anderlotinib. Sunitinib is a non-limiting example of a TKI that targetsreceptors for FGF, PDGF, and VEGF.

Diagnostic Methods

Over-expression of claudin 18.2 is observed in certain tumor samples,and patients having claudin 18.2-over-expressing cells are likelyresponsive to treatments with the anti-claudin 18.2 antibodies of thepresent disclosure. Accordingly, the antibodies of the presentdisclosure can also be used for diagnostic and prognostic purposes.

A sample that preferably includes a cell can be obtained from a patient,which can be a cancer patient or a patient desiring diagnosis. The cellbe a cell of a tumor tissue or a tumor block, a blood sample, a urinesample or any sample from the patient. Upon optional pre-treatment ofthe sample, the sample can be incubated with an antibody of the presentdisclosure under conditions allowing the antibody to interact with aclaudin 18.2 protein potentially present in the sample. Methods such asELISA can be used, taking advantage of the anti-claudin 18.2 antibody,to detect the presence of the claudin 18.2 protein in the sample.

Presence of the claudin 18.2 protein in the sample (optionally with theamount or concentration) can be used for diagnosis of cancer, as anindication that the patient is suitable for a treatment with theantibody, or as an indication that the patient has (or has not)responded to a cancer treatment. For a prognostic method, the detectioncan be done at once, twice or more, at certain stages, upon initiationof a cancer treatment to indicate the progress of the treatment.

Compositions

The present disclosure also provides pharmaceutical compositions. Suchcompositions comprise an effective amount of an antibody, and anacceptable carrier. In some embodiments, the composition furtherincludes a second anticancer agent (e.g., an immune checkpointinhibitor).

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans.Further, a “pharmaceutically acceptable carrier” will generally be anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents such as acetates,citrates or phosphates. Antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; and agents forthe adjustment of tonicity such as sodium chloride or dextrose are alsoenvisioned. These compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences by E. W. Martin, incorporated herein byreference. Such compositions will contain a therapeutically effectiveamount of the antigen-binding polypeptide, preferably in purified form,together with a suitable amount of carrier so as to provide the form forproper administration to the patient. The formulation should suit themode of administration. The parental preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

In an embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the disclosure can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

EXAMPLES Example 1: Generation of Murine Monoclonal Antibodies AgainstHuman Claudin 18 Isoform 2 (CLD 18A2)

a. Immunizations:

Balb/c and C57/BL6 mice were immunized with eukaryotic expressionvectors, encoding human claudin 18.2 (CLD 18A2) fragments. 50 μg ofplasmid DNA was injected into the quadriceps (intramuscular, i.m.) onday 1 and 10. The presence of antibodies directed against human CLD 18A2in the serum of the mice was monitored on day 20 by flow cytometry,using HEK293 cells transiently transfected with a nucleic acid encodinghuman CLD 18A2. Mice with detectable immune responses (FIG. 1) wereboosted three and two days prior to fusion by intraperitoneal injectionof 5×10⁷ HEK293 cells transiently transfected with a nucleic acidencoding human CLD 18A2.

b. Generation of Hybridomas Producing Human Monoclonal Antibodies toCLD18A2:

Mouse splenocytes were isolated and fused with PEG to a mouse myelomacell line based on standard protocols. The resulting hybridomas werethen screened for production of immunoglobulins with CLD 18A2specificity using HEK293 cells transfected with a nucleic acid encodinghuman CLD18 by cell ELISA.

Single cell suspensions of splenic lymphocytes from immunized mice werefused with P3×63Ag8U.1 non-secreting mouse myeloma cells (ATCC, CRL1597) in a 2:1 ratio using 50% PEG (Roche Diagnostics, CRL 738641).Cells were plated at approximately 3×10⁴/well in flat bottom microtiterplates, followed by about two weeks incubation in selective mediumcontaining 10% fetal bovine serum, 2% hybridoma fusion and cloningsupplement (HFCS, Roche Diagnostics, CRL 1 363 735) plus 10 mM HEPES,0.055 mM 2-mercaptoethanol, 50 μg/ml gentamycin and 1×HAT (Sigma, CRLH0262). After 10 to 14 days individual wells were screened by Cell ELISAfor anti-CLD 18A2 monoclonal antibodies (FIG. 2). The antibody secretinghybridomas were re-plated, screened again with HEK293 expressing CLD18A2 or CLD 18A1 by FACS and, if still positive for CLD18A2 and negativefor CLD18A1, were subcloned by limiting dilution. The stable subcloneswere then cultured in vitro to generate small amounts of antibody intissue culture medium for characterization. At least one clone from eachhybridoma, which retained the reactivity of parent cells (by FACS), waschosen. Three vial cell banks were generated for each clone and storedin liquid nitrogen.

c. Selection of Monoclonal Antibodies Binding to CLD 18A2 not to forCLD18A1:

To determine the isotype of antibodies, an isotype ELISA was performed.The mouse monoAB ID Kit (Zymed, CRL 90-6550) was used to determine Igsubclasses of the identified CLD18A2 reactive monoclonal antibodies.Thirty-two hybridoma cell lines were generated: 64G11B4, 65G8B8,56E8F10F4, 54A2C4, 44F6B11, 15C2B7, 20F1E10, 72C1B6A3, 58G2C2, 101C4F12,103A10B2, 40C10E3, 78E8G9G6, 4F11E2, 10G7G11, 12F1F4, 78C10B6G4,119G11D9, 113G12E5E6, 116A8B7, 105F7G12, 84E9E12, 103F4D4, 110C12B6,85H12E8, 103H2B4, 103F6D3, 113E12F7, 120B7B2, 111B12D11, 111E7E2, and100F4G12, with further details as shown below:

64G11B4, mouse monoclonal IgG1, κ antibody

65G8B8, mouse monoclonal IgG1, κ antibody

56E8F10F4, mouse monoclonal IgG1, κ antibody

54A2C4, mouse monoclonal IgG1, κ antibody

44F6B11, mouse monoclonal IgG1, κ antibody

15C2B7, mouse monoclonal IgG1, κ antibody

20F1E10, mouse monoclonal IgG1, κ antibody

72C1B6A3, mouse monoclonal IgG1, κ antibody

58G2C2, mouse monoclonal IgG2a, κ antibody

101C4F12, mouse monoclonal IgG2b, κ antibody

103A10B2, mouse monoclonal IgG2b, κ antibody

40C10E3, mouse monoclonal IgG1, λ antibody

78E8G9G6, mouse monoclonal IgG1, κ antibody

4F11E2, mouse monoclonal IgG1, κ antibody

10G7G11, mouse monoclonal IgG1, κ antibody

12F1F4, mouse monoclonal IgG1, κ antibody

78C10B6G4, mouse monoclonal IgG1, κ antibody

119G11D9, mouse monoclonal IgG1, κ antibody

113G12E5E6, mouse monoclonal IgG1, κ antibody

116A8B7, mouse monoclonal IgG1, κ antibody

105F7G12, mouse monoclonal IgG1, κ antibody

84E9E12, mouse monoclonal IgG1, κ antibody

103F4D4, mouse monoclonal IgG1, κ antibody

110C12B6, mouse monoclonal IgG1, κ antibody

85H12E8, mouse monoclonal IgG1, κ antibody

103H2B4, mouse monoclonal IgG1, κ antibody

103F6D3, mouse monoclonal IgG1, κ antibody

113E12F7, mouse monoclonal IgG2a, κ antibody

120B7B2, mouse monoclonal IgG2a, κ antibody

111B12D11, mouse monoclonal IgG2a, κ antibody

111E7E2, mouse monoclonal IgG2a, κ antibody

100F4G12, mouse monoclonal IgG3, κ antibody.

Example 2. Hybridoma Sequencing

Hybridoma cells (1×10⁷) were harvested and total RNA was extracted usingTri Reagent as described above for spleen tissue. cDNA was preparedusing SuperScript III kit according to the manufacturer's instruction,described above. The resulting cDNA product was used as template for PCRwith primers VhRevU and VhForU, the resulting 300 bp PCR product wascleaned up using a PCR clean-up kit and sequenced with the same primer.PCR reaction was also performed with light chain V-region specificprimer VkRev7 and VkFor (for variable region only) or KappaFor primers(for entire kappa light chain). Sequencing reactions were performed oncleaned PCR product to obtain DNA sequences for the antibodies, 64G11B4,65G8B8, 56E8F10F4, 54A2C4, 44F6B11, 15C2B7, 20F1E10, 72C1B6A3, 58G2C2,101C4F12, 103A10B2, 40C10E3, 78E8G9G6, 4F11E2, 10G7G11, 12F1F4,78C10B6G4, 119G11D9, 113G12E5E6, 116A8B7, 105F7G12, 84E9E12, 103F4D4,110C12B6, 85H12E8, 103H2B4, 103F6D3, 113E12F7, 120B7B2, 111B12D11,111E7E2, and 100F4G12. Their variable (VH and VL) sequences are shown inTable 1 below.

TABLE 1 Sequences of the variable regions of the antibodies Antibodychain Sequence SEQ ID NO: Light chains 64G11B4DIVMTQSPSSLTVTAGEKVTMNCKSSQSLLNSGNQRNYLTWYQQKPGQPP 117KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQADDLAVYYCQNDYFY PFTFGAGTNLELK 65G8B8DIMMTQSPSSLTVTTGEKVTLTCKSSQSLLNSGNLKNYLTWYQQKPGHPP 118KLLIYWASTRESGVPVRFTGSGSGTDFTLTISSVQAEDLTVYYCQNVYIY PFTFGSGTKLEMR56E8F10F4 DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPP 119KLLIYWASTRESGVPDRFTGSGSGTYFTLTISSVQAADLAVYYCQNDYYF PFTFGSGTKLEIK 54A2C4DTVMTQFPSSLSVSAGEKVTMSCKSSQSLLNGGNQKNYLAWYQQKPGQPP 120KLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDLYY PWTFGGGTKLEFK 44F6B11DIVMTQSPSSLTVTAGEKVIMSCKSNQSLLNSGNQKKYLTWYQQKPGQSP 121KLLIYWASTRESGVPDRFTGSESGTDFTLTISSVRAEDLAVYYCQNGYSY PFTFGSGTKLEMK 15C2B7DIVMTQSPSSLTVTAGGKVTVSCKSSQSLLNSGNQKNYLTWYQQKPGQPP 122KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQTEDLAVYYCQNNYYF PLTFGAGTKLELK 20F1E10DIVMTQSPSSLTVTAGEKVTMSCKSSQSLFNSGNQRNYLTWYQQKPGQPP 123KLLIYWASTRESGVPDRFTGSGSGTDFILTITKVQAEDLAVYYCQNVYSY PLTFGAGTKLELK72C1B6A3 DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQRPGQPP 124KLLIYRASSRESGVPVRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYIY PYTFGGGTKLEMN 58G2C2DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPP 125TLLIFWAFTRESGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCQNSYSY PFTFGSGTKLEIK101C4F12 DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQRNYLTWYQQKPGQPP 126RLLIYWSSTRDSGVPDRFTGSGSRTDFTLTISSVQAEDLAVYYCQNNFIY PLTFGAGTKLELK 103B2DIVMTQSPSSLTVTPGEKVTMSCRSSMSLFNSGNQKSYLSWYHQKPGQPP 127KLLIYWASTRDSGVPVRFTGSGSGTDFTLTISSVQAEDLAVYYCHNDYIY PLTFGAGTKLELK78E8G9G6 DIVMTQSPSSLTVTAGEKVTMNCRSIQSLLNSGNQKNYLSWYQQKPGQPP 128KLLIYWASTRESGVPDRFTGSGSGTDFTLTIRSVLDEDLAVYYCQNSYSY PFTFGSGTKLEMK 4F11E2DIVMTQSPSSLTVTAGEKVTLTCRSSQSLLNSGNRKNYLTWYQQIPGQPP 129KLLIYWASTRESGVPDRFTGSGSGTYFTLTISSVQAEDLAVYYCQNAYSY PFTFGSGTKLEKK 10G7G11DIVMTQSPSSLTVTAGEKVTMTCKSSQSLFNSGNQRNYLTWYQRKPGQPP 130KLLIYWASTRESGVPDRFTGSGSGTYFTLTVSSVQAEDLAVYYCQNAYYF PFTFGSGTKLEKK 12F1F4DIVMTQSPSSLTVTARERVSMTCKSSQSLFNSGNQRNYLTWYQQKPGQPP 131KLLIYWSSTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYFCQNNYYY PFTFGSGTKLEIK78C10B6G4 DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQRPGQPP 124KLLIYRASSRESGVPVRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTY PYTFGGGTKLEMN119G11D9 DIVMTQSPSSLTVTAGERVTMRCRSTQSLFNSGNQKNYLTWYQQKPGQPP 132KLLIYWASTRESGVPDRFTGGGSGTDFTLTISSVQAEDLAVYYCQNAYYY PLTFGAGTKLERK113G12E5E6 DIVMTQSPSSLTVTAGERVTMSCKPSQSLLNSGNQKNYLAWYQQKPGQPP 133KLLLYWASTRESGVPDRFKGSGSGTDFTLTISSVQAEDLAVYYCQNAYFY PCTFGGGTKLEMK 116A8B7DIVMTQSPSSLTVTAGEKVTMRCRSTQSLFNSGNQRNYLTWYQQKPGQPP 134KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNAYYY PLTFGVGTKLERK105F7G12 DIVMTQSPSSLTVTAGERVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPP 135KLLLYWASTRESGVPDRFKGSGSGTDFTLTISSVQAEDLAVYYCQNAYFY PCTFGGGTKLEMK 84E9E12DIVMTQSPSSLTVTTGEKVTMSCKSSQSVFNSGNQKNYLTWYQQKPGQPP 136KLLVYWASTRESGVPARFTGSGSGTVFTLTISSVQAEDLAVYYCQNDYYF PLTFGAGTRLELK 103F4D4DIVMTQSPSSQTVTAGEKVTLSCRSSQSLLNGGNQKNYLTWYQQKPGQPP 137KLLIYWASTRESGVPDRFTGSGSGTYFTFTISSVQAEDLAVYYCQNAYFY PFTFGAGTKLELK110C12B6 DIVMTQSPSSLTVTAGEKVTMRCRSTQSLFNSGNQRNYLTWYQQKPGQPP 134KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNAYYY PLTFGVGTKLERK 85H12E8DIVMTQSPSSLTVTAGEKVTMNCKSSQSLLNSGNQRNYLSWYQQEPGQPP 138KLLIYWASTRESGVPDRFTGSGSGTDFTLTISNIQAEDLALYFCQNAYFY PFTFGSGTKLEIK 103H2B4DILMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQSP 139KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNNYFY PLTFGVGTKLELK 103F6D3DIVMTQSPSSQTVTAGEKVTLSCRSSQSLLNGGNQKNYLTWYQQKPGQPP 137KLLIYWASTRESGVPDRFTGSGSGTYFTFTISSVQAEDLAVYYCQNAYFY PFTFGAGTKLELK113E12F7 DIVMTQSPSSLTVTTGEKVTMSCKSSQSLFNSGNQKNYLTWYQQKPGQPP 140KLLIYWASTRESGVPDRFTGSGSGTYFTLTISSVQAEDLAVYYCQNNYTY PLAFGTGTKLELK 120B7B2DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQRPGQPP 141KLLMYWASTRESGVPDRFTGSGSGTDFTLTISSGQAEDLAIYFCQNGYYF PFTFGSGTKLETK111B12D11 DIVMTQSPSSLTVTAGEKVTMRCRSSQSLFNSGNQRNYLTWYQQKPGQPP 142KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNNYTY PLAFGAGTKLELK 111E7E2DIVMTQSPSSLTVTAGEKVTMSCKSSQSLFNSGNQKNYLTWYQQKPGQPP 143KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNNYIY PLAFGAGTKLELK100F4G12 DIVMTQSPSSLTVTAGEKVTMRCKSTQSLLNSGNQRNYLTWYQQKPGQSP 144KLLIYWASTRESGVPERFTGSGSGTDFTLTISSVQAEDLAVYYCQNAYYY PLTFGPGTKLERKHeavy Chain 64G11B4 QVQLHQSGTELVRPGTSVKVSCKASGYAFTNYLLEWVKQRPGQGLEWIGE145 INPGNGGSNYNEKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARIYYGNSFAYWGQGTLVTVSA 65G8B8QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPPGKGLEWLGV 146IWGDGNTIYHSALKSRLSISRDNSKRQVFLKVNSLQIDDTATYYCAKQGL YGHAMDYWGQGTSVIVSS56E8F10F4 DVQLVESGGGLVQPGGSRKLSCTASGFTFNSFGMNWVRQAPEKGLEWVAF 147ISGGSNTIHYLDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCTRLA LGNAMDYWGQGTSVIVSS54A2C4 EVQHVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVAR 148IRSKSNNYATYYADSVKDRFTISRDDSQSMLYVQMNNLKTEDTAMYYCVSGAYYGNSKAFDYWGQGTLVTVSA 54A2C4′QVQLHQSGTELVRPGTSVKVSCKASGYAFTNYLLEWVKQRPGQGLEWIGE 145INPGNGGSNYNEKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARIY YGNSFAYWGQGTLVTVSA54A2C4″ QVQLQQSGAELARPGASVKMSCKASGYTFPTYSIHWLKQGPGQGLEWIGY 149INPSTIYTNYNQKFKYKATLTADKSSSTAYIQLSSLTSDDSAVYYCAREG YGRGNAMDYWGQGTSVTVSS44F6B11 EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTPDKRLEWVAT 150FSYGDSHNYYSDSVKGRFTISRDIAKDALYLQMSSLRSEDTAIYYCARFG RGNTMDYWGQGTSVTVSL15C2B7 QIQLVQSGPELRKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMAW 151INANTGEPTYAEEFKGRFAFSLETSARSAYLQINSLKNEDTATYFCARLT RGNSFDYWGQGTTLTVSS20F1E10 QIQLVQSGPELKKPGETVKISCKASGYTFTKYGMNWVRQAPGKGLKWMGW 152ISTNTGEPTYAEEFKGRFAFSLETSASTAFLQINNLKNEDTATYFCARLV RGNSFDFWGQGITLTVSS72C1B6A3 QVQLQQSGGELVKPGASVKMSCKAFGYTFTTYPIEWMKQNHGKSLEWIGN 153FHPYNDDTKYNEKFKGKAKLTVEKSSSTVYLEVSRLTSDDSAVYYCARRA YGYPYAMDYWGQGTSVTVSS58G2C2 QVHLQQSGAEVVRPGTSVKVSCKASGYAFTNYLIEWIKKRPGQGLEWVGV 154INPGRSGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARTR YGGNAMDYWGQGTSVTVSS101C4F12 QVQLKESGPGQVAPSQSLSIACTVSGFSLSSYGVHWVRQPPGKGLEWLGV 155IWAGGSTNYDSALMSRLTISKDNSRTRVFLKMNSLQTDDTAIYYCARSLY GNSLDSWGPGTTLTVSS103B2 QVQLKESGPGLVAPSQSLSITCTVSGLSLTSFGVHWIRQPPGKGLEWLGV 156IWAGGSTNYNSALMSRLSISKDNSKSQVYLKMHSLQTDDTAMYYCARSLY GNSFDYWGQGTALTVSS40C10E3 QVQLKESGPGLVAPSQSLSITCTVSGFSLSSYGVNWVRQPPGKGLEWLAA 157IRSDGIITYNSVLKSRLRISKDNSKSQVFLKMNSLQTDDTAMFYCARWFR GNVLDYWGQGTSVTVSS78E8G9G6 QVQLKESGPGLVAPSQSLSITCTVSGFSLISYGVHWVRQPPGKGLEWLGV 158IWAGGRTNYNSALMSRLSISKDNSKSQVFLKMNSLQTYDTAMYYCARDRY GGNSLDYWGQGTSVTVSS4F11E2 DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAY 159ITSGNSPIYFTDTVKGRFTISRDNPKNTLFLQMTSLGSEDTAVYYCARSS YYGNSMDYWGQGTSVTVSS10G7G11 QVQLKESGPGLVAPSQSLSITCTISGFSLNTYGVHWVRQPPGKGLEWLVV 160MLSDGNTVYNSSLKSRLSLTKDNSKSQLLLKMNSLQTDDTAIYYCARHKA YGNAMDYWGQGTSVTVSS12F1F4 QVQLKESGPGLVAPSQSLSITCTVSGFSLINYGVSWVRQPPGKGLEWLGV 161IWGDGNTNYQSALRSRLSIRKDTSKSQVFLKLNSVHTDGTATYYCAKVGR GNAMDHWGQGISVIVSS78C10B6G4 QVQLKESGPGLVAPSQSLSITCTVSGFSLINYGVSWVRQPPGKGLEWLGV 162IRGDGNTNYQSALRSRLSIRKDTSKSQVFLKLNSVHTDGTATYYCAKVGR GNAMDHWGQGISVIVSS119G11D9 EVQLQQSGPELVKPGASVKMSCKASGYTFTGFLMHWVKQKPGQGLEWIGY 163INPYNDGTKYSEKFKGKATLTSDKSSSTAFMELSSLTSDDSAVYYCARLD YGNAMDYWGQGTSVTVSS113G12E5E6 QVQLKQSGPGLVQPSQSLSITCTVSDFSLTKYGVHWFRQSPGKGLEWLGV 164IWTGGNTDYNPALIPRLSFRKDNSKSQVFFKMNSLQSSDTAVYYCARNGY YGNAMDYWGQGTSVTVSS116A8B7 EVQLQQSGPELVKPGASVKMSCKSSGYTFTGFLMHWVKQKPGQGLEWIGY 165INPYNDGTKYSEKFKGKATLTSDKSSSTAYMELSSLTSDDSAVYYCGRLD YGNAMDYWGQGTSVTVSS105F7G12 QVQLKQSGPGLVQPSQSLSITCTVSDFSLTKYGVHWFRQSPGKGLEWLGV 164IWTGGNTDYNPALIPRLSFRKDNSKSQVFFKMNSLQSSDTAVYYCARNGY YGNAMDYWGQGTSVTVSS84E9E12 DVQLQESGPGLVKPSQSLSLSCSVTGYSITSGYFWTWFRQFPGNKLEWMG 166YISYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCASFR FFAYWGQGTLVTVSA103F4D4 QVQLQQSGAELARPGASVKMSCKASGYTFPTYSIHWLKQGPGQGLEWIGY 149INPSTIYTNYNQKFKYKATLTADKSSSTAYIQLSSLTSDDSAVYYCAREG YGRGNAMDYWGQGTSVTVSS110C12B6 EVQLQQSGPELVKPGASVKMSCKSSGYTFTGFLMHWVKQKPGQGLEWIGY 165INPYNDGTKYSEKFKGKATLTSDKSSSTAYMELSSLTSDDSAVYYCGRLD YGNAMDYWGQGTSVTVSS85H12E8 QVQLKESGPGLVAPSQSLSITCTVSGFSLSNYGVSWVRQPPGKGLEWLGV 167IWAGGNTNYNSALMSRLRISKDNSKSQVFLKMNSLQTDDTARYYCARHGY GKGNAMDNWGQGTSVTVSS103H2B4 QVQLQQPGAEPVKPGASVKLSCKASGYSFTNFLTHWVRQRPGQGLEWIGE 168INPTNGRTYYNEKFKRKATLTVDKSSTTVYMQLSNLTPEDSAVFYCARIY YGNSMDYWGQGTLVTVSA103F6D3 QVQLQQSGAELARPGASVKMSCKASGYTFPTYSIHWLKQGPGQGLEWIGY 169INPNTIYTNYNQKFKYKTTLTADKSSSTAYIQLSSLTSDDSAVYYCAREG YGRGNAMDYWGQGTSVTVSS113E12F7 QVQLKESGPGLVAPSQSLSITCTVTGFSLSSYGVHWVRQPPGKGLEWLGV 170IWAGGSTNYDSALMSRLSISKDRSKSQVFLKMTSLQTDDTAMYYCARSLY GNSFDHWGQGTTLTVSS120B7B2 EVQLQQSGPELVKPGASVKMSCKASGYTFTGYIIQWMKQKPGLGLEWIGF 171INPYNDGTKYNEQFKGKATLTSDKSSNAAYMELSSLTSEDSAVYYCARAY FGNSFAYWGQGTLVTVSA111B12D11 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGV 172IWAGGSTNYDSTLMSRLSISKDRSKSQVFLKMTSLQTDDTAMYYCARSLY GNSFDHWGQGTTLTVSS111E7E2 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGAHWVRQPPGKGLEWLGV 173IWAGGSTNYDSALMSRLSISKDRSKSQVFLKMTSLQTDDTAMYYCARSLY GNSFDHWGQGTTLTVSS100F4G12 EVQLQQSGPELVKPGASVKMSCKASGYTFTGFLMHWVKQKPGQGLEWIGY 174INPYNDGTKYSERFKGKATLTSDKSSSTAYMELSSLTSDDSAVYYCARLD YGNAMDYWGQGTSVTVSS

Example 3. Production and Purification of Monoclonal Antibodies Reactiveto CLD18A2

To produce mg amounts of antibody for functional characterization,hybridoma cells were seeded in dialysis based bioreactors (CELLineCL1000, Integra, Chur, CH) at 2×10⁶ cells/ml. Antibody containingsupernatant was harvested once weekly. Mouse monoclonal antibody waspurified using Melon Gel (Pierce, Rockford, USA) and concentrated byammonium sulphate precipitation. Antibody concentration and purity wasestimated by sodium dodecylsulphate gel electrophoresis and coomassiestaining.

Example 4. Binding of Murine Monoclonal Antibodies Reactive to CLD18A2

MKN45 cells over-expressed CLD18A2 were harvested from flasks. 100 μl of1×10⁶ cells/ml of cells were incubated with primary antibodies indicatedas FIG. 3 in 3-fold serial dilutions starting from 100 nM to 0.003 nMfor 30 minutes on ice. After being washed with 200 μl of FACS buffertwice, cells were incubated with secondary antibody for 30 minutes onice. Cells were washed with 200 μl of FACS buffer twice and transferredto BD Falcon 5 ml tube and analyzed by FACS. The results of the studyshowed that the purified murine antibody could bind to MKN45 cellstransfected with human CLD18A2 by flow cytometry with high EC50,compared with positive reference antibody.

Example 5. Binding of Murine Monoclonal Antibodies Reactive to CLD18A2Mutant

SU620 cells that endogenously expressed CLD18A2 bearing M149L mutationwere harvested from flasks. 100 μl of 1×10⁶ cells/ml of cells wereincubated with primary antibodies indicated as FIG. 4 in 3-fold serialdilutions starting from 100 nM to 0.003 nM for 30 minutes on ice. Afterbeing washed with 200 μl of FACS buffer twice, cells were incubated withsecondary antibody for 30 minutes on ice. Cells were washed with 200 μlof FACS buffer twice and transferred to BD Falcon 5 ml tube and analyzedby FACS. The results of the study showed that the purified murineantibody could bind to SU620 cells endogenously expressing human CLD18A2bearing M149L mutation by flow cytometry with high EC50, while therefrence antibody did not (FIG. 4).

Example 6. Binding of Murine Monoclonal Antibodies Reactive to Mouse andCyno CLD18A2

To evaluate these antibodies cross-reactivities with mouse and cynoCLD18A2, HEK293 cells over-expressed mouse, cyno or human CLD18A2 wereharvested from flasks. 100 μl of 1×10⁶ cells/ml of cells were incubatedwith primary antibodies indicated as FIG. 3a in 3-fold serial dilutionsstarting from 100 nM to 0.003 nM for 30 minutes on ice. After beingwashed with 200 μl of FACS buffer twice, cells were incubated withsecondary antibody for 30 minutes on ice. Cells were washed with 200 μlof FACS buffer twice and transferred to BD Falcon 5 ml tube and analyzedby FACS. The results of the study showed that the purified murineantibodies can bind to mouse and cyno CLD18A2 by flow cytometry withhigh EC50, at least like the reference anbibodies (FIGS. 5, 6 and 7).

Example 7 Binding of Chimeric Antibodies Reactive to CLD18A2

The murine VH and VK genes were produced synthetically and thenrespectively cloned into vectors containing the human gamma 1 and humankappa constant domains. The purified chimeric antibodies were producedfrom transfected CHOs cells.

MKN45 cells that stably expressed human CLD18A2 or CLD18A1, wereharvested from flasks. 100 μl of 1×10⁶ cells/ml of cells were incubatedwith primary chimeric antibodies indicated as FIG. 4 in 3-fold serialdilutions starting from 100 nM to 0.003 nM for 30 minutes on ice. Afterbeing washed with 200 μl of FACS buffer twice, cells were incubated withsecondary antibody for 30 minutes on ice. Cells were washed with 200 μlof FACS buffer twice and transferred to BD Falcon 5 ml tube and analyzedby FACS. The results of the study showed that the chimeric antibodiescan bind to human CLD18A2 with high EC50, while not CLD18A1 (FIGS. 8 and9).

Example 8. Antibody-Dependent Cellular Cytotoxicity (ADCC) of ChimericAntibodies

The ADCC Reporter Bioassay uses an alternative readout at an earlierpoint in ADCC MOA pathway activation: the activation of genetranscription through the NFAT (nuclear factor of activated T-cells)pathway in the effector cell. In addition, the ADCC Reporter Bioassayuses engineered Jurkat cells stably expressing the FcγRIIIa receptor,V158 (high affinity) variant, and an NFAT response element drivingexpression of firefly luciferase as effector cells. Antibody biologicalactivity in ADCC MOA is quantified through the luciferase produced as aresult of NFAT pathway activation; luciferase activity in the effectorcell is quantified with luminescence readout (FIG. 1). Signal is high,and assay background is low.

Serial dilutions of claudin 18.2 chimeric monoclonal antibody or Ref. Abwere incubated for 6 hours of induction at 37° C. with engineered Jurkateffector cells (ADCC Bioassay Effector Cells), with or without ADCCBioassay Target Cells (expressing claudin 18.2). Luciferase activity wasquantified using Bio-Glo™ Reagent (Table 2). The results show that thesechimeric antibodies have very strong ADCC activities.

TABLE 2 EC50 of the tested antibodies EC50 Antibody (pM) 4F11E2 22.1812F1F4 36.77 64G11B4 125.7 72C186A3 46.32 78E8G9G6 15.86 103F6D3 79.53120B7B2 5.806 Ref. Ab 458.5

Example 9. Humanization of the 4F11E2, 72C1B6A3 and 120B7B2 Mouse mAbs

The mAb 4F11E2, 72C1B6A3 and 120B7B2 variable region genes were employedto create humanized MAbs. In the first step of this process, the aminoacid sequences of the VH and VL of MAb were compared against theavailable database of human Ig gene sequences to find the overallbest-matching human germline Ig gene sequences.

The amino acid sequences of the humanized antibodies are listed in Table3 below.

TABLE 3 Humanized sequences Name Sequence SEQ ID NO: 4F11VH_1EVQLVESGGGLVQPGGSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWVSY 175 (grafted VH)ITSGNSPIYFTDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSS YYGNSMDYWGQGTLVTVSS4F11VH_2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWVAY 176ITSGNSPIYFTDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSS YYGNSMDYWGQGTLVTVSS4F11VH_3 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWVAY 177ITSGNSPIYFTDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARSS YYGNSMDYWGQGTLVTVSS4F11VL_1 DIVMTQSPDSLAVSLGERATINCRSSQSLLNSGNRKNYLTWYQQKPGQPP 178(grafted VL) KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYSYPFTFGGGTKLEIK 4F11VL_2DTVMTQSPDSLAVSLGERATINCRSSQSLLNSGNRKNYLTWYQQKPGQPP 179KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQNAYSY PFTFGGGTKLEIK4F11VL_3 DTVMTQSPDSLAVSLGERVTLNCRSSQSLLNSGNRKNYLTWYQQKPGQPP 180KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQNAYSY PFTFGGGTKLEIK72C1 VH_ 1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYPIEWVRQAPGQRLEWMGN 181(grafted VH) FHPYNDDTKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARRAYGYPYAMDYWGQGTLVTVSS 72C1VH_2QVQLVQSGAEVVKPGASVKVSCKASGYTFTTYPIEWMRQAPGQRLEWMGN 182FHPYNDDTKYNEKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARRA YGYPYAMDYWGQGTLVTVSS72C1VH_3 QVQLVQSGAEVVKPGASVKVSCKASGYTFTTYPIEWMKQAPGQRLEWMGN 183FHPYNDDTKYNEKFKGRVTITVDTSASTAYMEVSSLRSEDTAVYYCARRA YGYPYAMDYWGQGTLVTVSS72C1VH_4 QVQLVQSGAEVVKPGASVKMSCKASGYTFTTYPIEWMKQAPGQRLEWMGN 184FHPYNDDTKYNEKFKGRVTLTVDTSASTVYLEVSSLRSEDTAVYYCARRA YGYPYAMDYWGQGTLVTVSS72C1VL_1 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPP 185(grafted VL) KLLIYRASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYIYPYTFGGGTKLEIK 72C1VL_2DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPP 186KLLIYRASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYIY PYTFGGGTKLEIK72C1VL_3 DIVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPP 187KLLIYRASSRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQNDYIY PYTFGGGTKLEIK120B7VH_1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYIIQWVRQAPGQRLEWMGF 188(grafted VH) INPYNDGTKYNEQFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAYFGNSFAYWGQGTLVTVSS 120B7VH_2QVQLVQSGAEVVKPGASVKVSCKASGYTFTGYIIQWMRQAPGQRLEWMGF 189INPYNDGTKYNEQFKGRVTITSDTSASAAYMELSSLRSEDTAVYYCARAY FGNSFAYWGQGTLVTVSS120B7VH_3 QVQLVQSGAEVVKPGASVKVSCKASGYTFTGYIIQWMKQAPGQRLEWIGF 190INPYNDGTKYNEQFKGRATITSDTSASAAYMELSSLRSEDTAVYYCARAY FGNSFAYWGQGTLVTVSS120B7VH_4 EVQLVQSGAEVVKPGASVKMSCKASGYTFTGYIIQWMKQAPGQRLEWIGF 191INPYNDGTKYNEQFKGRATLTSDTSASAAYMELSSLRSEDTAVYYCARAY FGNSFAYWGQGTLVTVSS120B7VL_1 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPP 192(grafted VL) KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNGYYFPFTFGGGTKLEIK 120B7VL_2DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPP 193KLLMYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNGYYF PFTFGGGTKLEIK120B7VL_3 DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPP 194KLLMYWASTRESGVPDRFSGSGSGTDFTLTISSGQAEDVAVYFCQNGYYF PFTFGGGTKLEIK120B7VL_4 DIVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPP 195KLLMYWASTRESGVPDRFSGSGSGTDFTLTISSGQAEDVAVYFCQNGYYF PFTFGGGTKLEIK

The humanized VH and VL genes were produced synthetically and thenrespectively cloned into vectors containing the human gamma 1 and humankappa constant domains. The pairing of the human VH and the human VLcreated the humanized antibodies (see Table 4).

TABLE 4 Humanized antibodies with their VH an VL regions 4F11E2 VHVL4F11VH_1 4F11VH_2 4F11VH_3 4F11VL_1 hu4F11.1 hu4F11.4 hu4F11.7 4F11VL_2hu4F11.2 hu4F11.5 hu4F11.8 4F11VL_3 hu4F11.3 hu4F11.6 hu4F11.9 72C1B6A3VHVL 72C1VH_1 72C1VH_2 72C1VH_3 72C1VH_4 72C1VL_1 hu72C1.10 hu72C1.13hu72C1.16 hu72C1.19 72C1VL_2 hu72C1.11 hu72C1.14 hu72C1.17 hu72C1.2072C1VL_3 hu72C1.12 hu72C1.15 hu72C1.18 hu72C1.21 120B7B2 VHVL 120B7VH_1120B7VH_2 120B7VH_3 120B7VH_4 120B7VL_1 hu120B7.22 hu120B7.26 hu120B7.30hu120B7.34 120B7VL_2 hu120B7.23 hu120B7.27 hu120B7.31 hu120B7.35120B7VL_3 hu120B7.24 hu120B7.28 hu120B7.32 hu120B7.36 120B7VL_4hu120B7.25 hu120B7.29 hu120B7.33 hu120B7.37

Example 10. Binding of Humanized Antibodies Reactive to CLD18A2

MKN45 cells that stably expressed human CLD18A2 or CLD18A1, wereharvested from flasks. 100 μl of 1×10⁶ cells/ml of cells were incubatedwith primary humanized antibodies indicated as FIG. 4 in 3-fold serialdilutions starting from 100 nM to 0.003 nM for 30 minutes on ice. Afterbeing washed with 200 μl of FACS buffer twice, cells were incubated withsecondary antibody for 30 minutes on ice. Cells were washed with 200 μlof FACS buffer twice and transferred to BD Falcon 5 ml tube and analyzedby FACS. The results of the study showed that the indicated humanizedantibodies can bind to human CLD18A2 with high EC50, while not CLD18A1(FIGS. 10 and 11).

Example 11. Binding of PTM (Post-Translational Modification) De-RiskHumanized Antibodies Reactive to CLD18A2

Post-translational modifications (PTMs) can cause problems during thedevelopment of a therapeutic protein such as increased heterogeneity,reduced bioactivity, reduced stability, immunogenicity, fragmentationand aggregation. The potential impact of PTMs depends on their locationand in some cases on solvent exposure. The CDRs of sequence wereanalyzed for the following potential PTMs: Asparagine deamidation,Aspartate isomerization, free Cysteine thiol groups, N-glycosylation,oxidation, fragmentation by potential hydrolysis site etc.

To reduce the risk of developing PTM in 4F11E2, 72C1B6A3 and 120B7B2,some concerned amino acids in the VH and VL were mutated. And then nineantibodies were generated:

Clones HC:LC* No. 4F11E2 HC N55Q-LC N31E 1 HC N55Q-LC S32A 2 HC N55E-LCS32A 3 HC N55E&N104Q-LC S32A 8 HC N55E&N104E-LC S32A 9 HC N55E&S105A-LCS32A 10 72C1B6A3 HC WT-LC N31E 4 HC WT-LC S32A 5 120B7B2 HCG57D&S104A-LC-N96E&N31E 6 HC G57D&S104A-LC-S32A&G97A 7 *The amino acidlocation (e.g., N55) is according to the amino acid residue number inthe corresponding VH or VL amino acid sequence, not Kabat or Chothia.

Antibody Mutant Sequence (mutation highlighted) SEQ ID NO: 4F11E2HC N55Q EVQLVESGGGLVQPGGSLRLSCAASGFITSTFGMHWVRQAPGKGLEWVSY 196 ITSG QSPIYFTDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSS YYGNSMDYWGQGTLVTVSSHC N55E EVQLVESGGGLVQPGGSLRLSCAASGFTESTEGMHWVRQAPGKGLEWVSY 197 ITSG ESPIYFTDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSS YYGNSMDYWGQGTLVTVSSHC N55E & EVQLVESGGGLVQPGGSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWVSY 198 N104QITSG E SPIYFTDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSS YYG QSMDYWGQGTLVTVSS HC N55E &EVQLVESGGGLVQPGGSLRLSCAASGFTESTEGMHWVRQAPGKGLEWVSY 199 N104E ITSG ESPIYFTDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSS YYG E SMDYWGQGTLVTVSSHC N55E & EVQLVESGGGLVQPGGSLRLSCAASGFITSTFGMHWVRQAPGKGLEWVSY 200 S105AITSG E SPIYFTDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSS YYGN AMDYWGQGTLVTVSS LC N31E DIVMTQSPDSLAVSLGERATINCRSSQSLL ESGNRKNYLTWYQQKPGQPP 201KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYSY PFTFGGGTKLEIK LC S32ADIVMTQSPDSLAVSLGERATINCRSSQSLLN A GNRKNYLTWYQQKPGQPP 202KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYSY PFTFGGGTKLEIK72C1B6A3 HC WT QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYPIEWVRQAPGQRLEWMGN 181FHPYNDDTKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARRA YGYPYAMDYWGQGTLVTVSSLC S32A DIVMTQSPDSLAVSLGERATINCKSSQSLLN A GNQKNYLTWYQQKPGQPP 203KLLIYRASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYIY PYTFGGGTKLEIK LC N31EDIVMTQSPDSLAVSLGERATINCKSSQSLL E SGNQKNYLTWYQQKPGQPP 204KLLIYRASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYIY PYTFGGGTKLEIK 120B7B2HC G57D & QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYIIQWVRQAPGQRLEWMGF 205 S104AINPYND D TKYNEQFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARAY FGN AFAYWGQGTLVTVSS LC-S32A & DIVMTQSPDSLAVSLGERATINCKSSQSLLN AGNQKNYLTWYQQKPGQPP 206 G97AKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQN A YYF PFTFGGGTKLEIKLC-N96E & DIVMTQSPDSLAVSLGERATINCKSSQSLL E SGNQKNYLTWYQQKPGQPP 207 N31EKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQ E GYYF PFTFGGGTKLEIK

MKN45 cells that stably expressed human CLD18A2 or CLD18A1 wereharvested from flasks. 100 μl of 1×10⁶ cells/ml of cells were incubatedwith primary mutated humanized antibodies indicated as FIG. 4 in 3-foldserial dilutions starting from 100 nM to 0.003 nM for 30 minutes on ice.After being washed with 200 μl of FACS buffer twice, cells wereincubated with secondary antibody for 30 minutes on ice. Cells werewashed with 200 μl of FACS buffer twice and transferred to BD Falcon 5ml tube and analyzed by FACS. The results of the study showed that theindicated antibodies can bind to human CLD18A2 with high EC50, while notCLD18A1 (FIGS. 12 and 13).

To evalute the de-risked variants of 4F11E2d (HC N55E/LC S32A) and4F11E2d (H N55E N104Q/LC S32A) antigen binding potency, the variantswere tested in a cell-based binding assay. Serially dilutedanti-CLDN18.2 antibodies, starting from 100 nM, were incubated with 10⁵cells for 30 minutes on ice. After being washed with FACS buffer, cellswere then incubated with APC labeled secondary antibody for another 30minutes on ice. Cells bound with antibodies were analyzed by FACS. Thevariants showed potent binding to cell surface claudin 18.2 (FIG. 14).

Example 12. Antibody-Dependent Cellular Cytotoxicity (ADCC) of PTMDe-Risked Humanized Antibodies

Serial dilutions of claudin 18.2 PTM de-risked humanized antibodies orRef. Ab were incubated for 6 hours of induction at 37° C. withengineered Jurkat effector cells (ADCC Bioassay Effector Cells), with orwithout ADCC Bioassay Target Cells (expressing claudin 18.2). Luciferaseactivity was quantified using Bio-Glo™ Reagent (Table 5). The resultsshow that these humanized antibodies have very strong ADCC activities.

EC50 No. Tested antibody (μM) 1 4E11E2-HC N55Q-LC N31E 238.1 2 4E11E2-HCN55Q-LC S32A 413.9 3 4E11E2-HC N55E-LCS32A 148.1 4 72C1B6A3-HC WT-LCN31E 1651 5 72C1B6A3-HC WT-LCS32A 190.5 6 120B7B2-HC G57D&104A- 492.6LC-N96E&N31E 7 120B7B2-HC G57D& 113.9 104A-LC-S32A&G97A Ref. Ab Ref. Ab158.3

Example 13. Epitope Mapping

All amino acids of extracellular domain of claudin 18.2 were singlemutated to A. The each mutated or wildtype claudin 18.2 was transfectedinto Hek293 cells. The expression of claudin 18.2 was evaluated byindicated antibodies. The results are shown in FIG. 14 (only amino acidresidues at which the mutation reduced bindings are shown).

As shown in FIG. 15, amino acids W30, N45, Y46, G48, L49, W50, C53, V54,R55, E56, S58, F60, E62, C63, R80, Y169, and G172 are involved in thebinding of the three tested antibodies, 4F11E2 (H4F), 72C186A3 (H72C1)and 120B7B2 (120), or the reference antibody 175D10 (IMAB362). W30appeared to form a cluster of residues at the first half of the firstextracellular domain of the claudin 18.2 protein. N45, Y46, G48, L49,W50, C53, V54, R55, E56, S58, F60, E62 and C63 appeared to be a secondcluster of residues within the same extracellular domain. Y169 and G172,on the other hand, are located at or close to the second extracellulardomain.

Example 14. Comparison of Humanized 4F11E2, 72C1B6A3 and 120B7B2Antibodies with Benchmark 175D10 Claudin 18.2 Antibody Cell BasedBinding

To compare the humanized anti-claudin18.2 antibodies: 4F11E2 (HC N55E/LCS32A), 72C1B6A3 (HC WT/LC S32A) and 120B7B2 (HC G57D S104A/LC S32A G97A)to benchmark antibody 175D10 (IMAB362), this example determined thecell-based binding in human claudin 18.2 expressed cells. CHO-K1 cellsthat stably expressed human CLD18A2 were sorted for high expressor andlow expressor based on the level of human CLDN18.2 expression. Seriallydiluted anti-CLDN18.2 antibodies, starting from 100 nM, were incubatedwith 10⁵ cells for 30 minutes on ice. After being washed with FACSbuffer, cells were then incubated with APC labeled secondary antibodyfor another 30 minutes on ice. Cells bound with antibodies were analyzedby FACS.

As shown in the FIG. 16, 4F11E2, 72C1B6A3 and 120B7B2 showed superiorbinding to 175D10 in both claudin 18.2 high and low CHO-K1 cells.

ADCC Assay

To further compare the ADCC effect of humanized anti-claudin18.2antibodies: 4F11E2 (HC N55E/LC S32A), 72C1B6A3 (HC WT/LC S32A) and120B7B2 (HC G57D S104A/LC S32A G97A) to benchmark antibody 175D10(IMAB362), this example performed a cell based ADCC assay. Briefly, NK92cells were cocultured with claudin18.2 overexpressed 293 cells in thepresence of different dose anti-claudin 18.2 antibodies. As shown inFIG. 17, 4F11E2, 72C1B6A3 and 120B7B2 showed superior ADCC potency tothe 175D10 antibody.

For certain therapeutic antibodies, enhanced ADCC may increase thetherapeutic window to antibody-based target therapy Enhanced ADCC may beachieved through engineering the Fc region such as with the S239D/I332Emutations. In a NK92 cell based ADCC assay, the 4H11E2, 72C1B6A3 and120B7B2 antibodies with S239D/I332E mutations in the Fc region mediatedstronger NK92-mediated cell killing of claudin 18.2 overexpressed 293cells as compared to the control antibody 175D10 with the sameS239D/I332E mutations (FIG. 18).

Antibody-Dependent Cellular Phagocytosis (ADCP)

The effect of anti-CLDN 18.2 mAbs on the tumor cell phagocytosis bymacrophages was evaluated in an in vitro assay in which CLDN18.2positive NUG-C4 cells were co-cultured with human differentiatedmacrophages in the presence of different concentration of anti-CLDN18.2mAbs. In short, CD14+ monocytes were purified from human peripheralblood mononuclear cells (PBMCs) and in vitro differentiated into maturemacrophages for 6 days. The monocyte derived macrophages (MDMs) werecollected and re-plated in 24-well dishes overnight as effector cells.NUG-C4 expressing CLDN 18.2-eGFP as target cells were added to MDMs at aratio of 5 tumor cells per phagocyte in the presence of differentconcentrations of anti-CLDN18.2 mAbs. After 3 hours' incubation,non-phagocytosed target cells were washed away with PBS and theremaining phagocytes were collected and stained with macrophage markerCD14 followed by flow cytometry analysis. Phagocytosis index wascalculated by quantitating the percent of GFP+ cells in CD14+ cells,normalized to that of IgG control.

As shown in FIG. 19, all C18.2 mAbs significantly enhanced thephagocytosis of NUG-C4 cells in a concentration-dependent manner In bothwildtype IgG1 and S239D/I332E mutated IgG1 formats, 4H11E2, 72C1B6A3 and120B7B2 antibodies showed stronger ADCP effect than the referenceantibody 175D10.

Taken together, this example demonstrates that the newly developed4F11E2, 72C1B6A3 and 120B7B2 antibodies had stronger cell-based bindingand ADCC/ADCP potency than the reference antibody 175D10. It iscontemplated that the improved properties of these new antibodies can beattributed to the higher binding specificity of these antibodies ascompared to that of the reference antibody 175D10. For instance,175D10's interaction with claudin 18.2 is strong across the spectrum inFIG. 15, which includes strong binding to D28, Q33, N38 and V43, andthen G59 and V79. The new antibodies, 4F11E2, 72C1B6A3 and 120B7B2, bycontrast, have higher specificity to W30 within the first half of thefirst extracellular domain, and higher specificity to G48 through E56within the second half of the first extracellular domain. The newantibodies also have slightly stronger binding to Y46 which is also inthe second half. Their binding to D28, Q33, N38, V43, G59 and V79 isconsiderably weaker, which likely contributed to the improved ADCC andADCP of the new antibodies.

The present disclosure is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the disclosure, and any compositions or methodswhich are functionally equivalent are within the scope of thisdisclosure. It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present disclosure without departing from the spirit or scope ofthe disclosure. Thus, it is intended that the present disclosure coverthe modifications and variations of this disclosure provided they comewithin the scope of the appended claims and their equivalents.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

What is claimed is:
 1. A method for treating cancer, comprisingadministering to a cancer patient an antibody or fragment thereof havingbinding specificity to a wild-type human claudin 18.2 (CLDN18.2)protein, wherein the binding between the antibody or fragment thereofand the wild-type CLDN18.2 protein involves amino acid residuescomprising: at least an amino acid residue selected from the groupconsisting of Y46, G48, L49, W50, C53, V54, R55, E56 and S58; and atleast an amino acid residue selected from the group consisting of Y169and G172, of the wild-type CLDN18.2 protein.
 2. The method of claim 1,wherein the binding further involves W30 of the CLDN18.2 protein.
 3. Themethod of claim 2, wherein the binding involves amino acid residuescomprising: W30; two or more amino acid residues selected from the groupconsisting of G48, L49, W50, C53, V54, R55, and E56; and at least anamino acid residue selected from the group consisting of Y169 and G172,of the wild-type CLDN18.2 protein.
 4. The method of claim 3, wherein thebinding involves amino acid residues comprising W30, G48, L49, W50, C53,E56 and Y169 of the wild-type CLDN18.2 protein.
 5. The method of claim1, wherein the binding involves at least Y46 or S58.
 6. The method ofclaim 1, wherein the binding does not include D28, Q33, N38, V43, G59and V79.
 7. The method of claim 1, which further binds to a M149L mutantof the CLDN18.2 protein at an affinity that is at least about 1% of theaffinity to the wild-type CLDN18.2 protein.
 8. The method of claim 7,which does not bind to a human wild-type claudin 18.1 (CLDN18.1) proteinat an affinity that is greater than about 1% of the affinity to thewild-type CLDN18.2 protein.
 9. The method of claim 1, wherein thepatient has a M149L mutant in the CLDN18.2 protein.
 10. The method ofclaim 1, wherein the antibody or fragment thereof comprises a lightchain variable region comprising light chain complementarity determiningregions CDRL1, CDRL2, and CDRL3 and a heavy chain variable regioncomprising heavy chain complementarity determining regions CDRH1, CDRH2,and CDRH3, and wherein: the CDRL1 comprises the amino acid sequence ofSEQ ID NO:216, 308 or 309, the CDRL2 comprises the amino acid sequenceof SEQ ID NO:227, the CDRL3 comprises the amino acid sequence of SEQ IDNO:13, the CDRH1 comprises the amino acid sequence of SEQ ID NO:246, theCDRH2 comprises the amino acid sequence of SEQ ID NO:268, 310 or 311,and the CDRH3 comprises the amino acid sequence of SEQ ID NO:294, 312,313 or
 314. 11. The method of claim 10, wherein the light chain variableregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:129, 178-180 and 201-202.
 12. The method ofclaim 11, wherein the heavy chain variable region comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:159,175-177 and 196-200.
 13. The method of claim 10, wherein: the CDRL1comprises the amino acid sequence of SEQ ID NO:309, the CDRL2 comprisesthe amino acid sequence of SEQ ID NO:227, the CDRL3 comprises the aminoacid sequence of SEQ ID NO:13, the CDRH1 comprises the amino acidsequence of SEQ ID NO:246, the CDRH2 comprises the amino acid sequenceof SEQ ID NO:311, and the CDRH3 comprises the amino acid sequence of SEQID NO:294.
 14. The method of claim 13, wherein the light chain variableregion comprises the amino acid sequence of SEQ ID NO:202 and the heavychain variable region comprises the amino acid sequence of SEQ IDNO:197.
 15. The method of claim 1, wherein the antibody or fragmentthereof further has a binding specificity to a second target protein.16. The method of claim 15, wherein the second target protein isselected from the group consisting of CD3, CD47, PD1, PD-L1, LAG3, TIM3,CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, CMET, 4-1BB,OX40, SIRPA CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, andVEGF.
 17. The method of claim 1, wherein the cancer is selected from thegroup consisting of bladder cancer, liver cancer, colon cancer, rectalcancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, smallcell lung cancer, non-small cell lung cancer, breast cancer, urethralcancer, head and neck cancer, gastrointestinal cancer, stomach cancer,oesophageal cancer, ovarian cancer, renal cancer, melanoma, prostatecancer and thyroid cancer.
 18. The method of claim 17, wherein thecancer is gastric cancer.